Composition for treating lung cancer, particularly of non-small lung cancers (nsclc)

ABSTRACT

The present invention relates to an active (immunostimulatory) composition comprising at least one RNA, preferably a mRNA, encoding at least two (preferably different) antigens capable of eliciting an (adaptive) immune response in a mammal. The invention furthermore relates to a vaccine comprising said active (immunostimulatory) composition, and to the use of said active (immunostimulatory) composition (for the preparation of a vaccine) and/or of the vaccine for eliciting an (adaptive) immune response for the treatment of lung cancer, particularly of non-small cell lung cancers (NSCLC), preferably selected from the three main sub-types squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma, or of disorders related thereto. Finally, the invention relates to kits, particularly to kits of parts, containing the active (immunostimulatory) composition and/or the vaccine.

The present invention relates to an active (immunostimulatory)composition comprising at least one RNA, preferably a mRNA, encoding atleast two (preferably different) antigens capable of eliciting an(adaptive) immune response in a mammal. The invention furthermorerelates to a vaccine comprising said active (immunostimulatory)composition, and to the use of said active (immunostimulatory)composition (for the preparation of a vaccine) and/or of the vaccine foreliciting an (adaptive) immune response for the treatment of lungcancer, particularly of non-small cell lung cancers (NSCLC), preferablyselected from the three main sub-types squamous cell lung carcinoma,adenocarcinoma and large cell lung carcinoma, or of disorders relatedthereto. Finally, the invention relates to kits, particularly to kits ofparts, containing the active (immunostimulatory) composition and/or thevaccine.

Of all malignant tumors 25% are bronchial carcinoma (carcinoma of thelung). Worldwide, it is the most common cause of cancer-related death inmen and the second most common in women. In Germany it is the third mostabundant sort of carcinoma following carcinoma of the prostata and thecolorectal carcinoma. It is responsible for 1.3 million deaths worldwideannually. In Central Europe the incidence is approximately 60 per100.000 inhabitants and the number of newly people diagnosed with lungcancer is steadily on the rise (in Germany currently being atapproximately 50.000 per year). When diagnosed with lung cancer theaverage overall fife-year survival rate is a mere 5 percent.Nevertheless, the life expectancy of each single patient is whollydependent on the disease stage (TMN classification) and the subtype ofcarcinoma (lung cancer) encountered (see below).

The main sub-types of lung cancer categorized by the size and appearanceof the malignant cells identified under microscope are small cell lungcancer (20%) and non-small cell lung cancer (NSCLC) (80%). Thisclassification, although based on simple histological criteria, has veryimportant implications for clinical management and prognosis of thedisease, with small cell lung cancer usually being treated bychemotherapy, while non-small cell lung cancer is mostly subject tosurgery as a first-line treatment.

The non-small cell lung cancers (NSCLC) are grouped together becausetheir prognosis and management are roughly identical. There are threemain sub-types: squamous cell lung carcinoma, adenocarcinoma and largecell lung carcinoma. Surgery is the mainstay of treatment; however, onlya quarter of the patients undergo successful resection, with arecurrence rate of 50%. Therapeutic approaches in advanced diseaseinvolve—following surgery—both adjuvant chemotherapy and/or adjuvantradiotherapy, whereas chemotherapy as monotherapy (first-line therapy)seems to be an approach associated with relatively poor results. In acomparison of four commonly used combination chemotherapy regimens, nonewas superior. Response rates varied from 15% to 22%, with 1-yearsurvival rates of 31% to 36% (see e.g. O'Mahony, D., S. Kummar, et al.(2005). “Non-small-cell lung cancer vaccine therapy: a concise review.”J Clin Oncol 23(35): 9022-8). Thus, even though preoperativechemotherapy seems to have not resulted in a prolongation of lifeexpectancy, adjuvant chemotherapy—also if combined with radiotherapy—didshow a significant increase in life expectancy.

One of the chemotherapeutic approaches used today are combinations ofplatin-based substances with e.g. Gemcitabin even as first-line-therapy,wheras e.g. Pemetrexed is used as second-line therapy.

Another option used for the treatment of NSCLC is the so-called“Targeted Therapy” trying to enhance success of classical cytotoxicchemotherapy by influencing tumor specific target structures on amolecular level. Substances used include Bevacizumab (an angiogenesisinhibitor) or Erlotinib, which is aimed at the tyrosine kinases of theepidermal growth factor receptor (EGFR).

Even though doubtless there is some improvement in the currenttherapeutic approaches treatment of lung cancer, especially of NSCLC, isstill an uphill-struggle with—given the high mortality rates—a strongneed for further, alternative or improved ways of treatment.

Thus, it is suggested here to use the immune system in an approach forthe treatment of the NSCLC. The immune system plays an important role inthe treatment and prevention of numerous diseases. According to thepresent stage of knowledge, various mechanisms are provided bymammalians to protect the organism by identifying and killing e.g. tumorcells. These tumor cells have to be detected and distinguished from theorganism's normal cells and tissues.

The immune system of vertebrates such as humans consists of many typesof proteins, cells, organs, and tissues, which interact in an elaborateand dynamic network. As part of this more complex immune response, thevertebrate system adapts over time to recognize particular pathogens ortumor cells more efficiently. The adaptation process createsimmunological memories and allows even more effective protection duringfuture encounters. This process of adaptive or acquired immunity formsthe basis for vaccination strategies.

The adaptive immune system is antigen-specific and requires therecognition of specific “self” or “non-self” antigens during a processcalled antigen presentation. Antigen specificity allows for thegeneration of responses that are tailored to specific pathogens orpathogen-infected cells or tumor cells. The ability to mount thesetailored responses is maintained in the body by so called “memorycells”. Should a pathogen infect the body more than once, these specificmemory cells are used to quickly eliminate it. The adaptive immunesystem thus allows for a stronger immune response as well as for animmunological memory, where each pathogen or tumor cell is “remembered”by one or more signature antigens.

The major components of the adaptive immune system in vertebratespredominantly include lymphocytes on the cellular level and antibodieson the molecular level.

Lymphocytes as cellular components of the adaptive immune system includeB cells and T cells which are derived from hematopoietic stem cells inthe bone marrow. B cells are involved in the humoral response, whereas Tcells are involved in cell mediated immune response. Both B cells and Tcells carry receptor molecules that recognize specific targets. T cellsrecognize a “non-self” target, such as a pathogenic target structure,only after antigens (e.g. small fragments of a pathogen) have beenprocessed and presented in combination with a “self” receptor called amajor histocompatibility complex (MHC) molecule. In contrast, the B cellantigen-specific receptor is an antibody molecule on the B cell surface,and recognizes pathogens as such when antibodies on its surface bind toa specific foreign antigen. This antigen/antibody complex is taken up bythe B cell and processed by proteolysis into peptides. The B cell thendisplays these antigenic peptides on its surface MHC class II molecules.This combination of MHC and antigen attracts a matching helper T cell,which releases lymphokines and activates the B cell. As the activated Bcell then begins to divide, its offspring secretes millions of copies ofthe antibody that recognizes this antigen. These antibodies circulate inblood plasma and lymph, bind to pathogens or tumor cells expressing theantigen and mark them for destruction by complement activation or foruptake and destruction by phagocytes.

As a cellular component of the adaptive immune system cytotoxic T cells(CD8⁺) may form a CTL-response. Cytotoxic T cells (CD8⁺) can recognizepeptides from endogenous pathogens and self-antigens bound by MHC type Imolecules. CD8⁺-T cells carry out their killing function by releasingcytotoxic proteins in the cell.

Mechanisms of the immune system form targets for curative treatments.Appropriate methods are typically based on the administration ofadjuvants to elicit an innate immune response or on the administrationof antigens or immunogens in order to evoke an adaptive immune response.As antigens are typically based on specific components of pathogens(e.g. surface proteins) or fragments thereof, administration of nucleicacids to the patient which is followed by the expression of desiredpolypeptides, proteins or antigens is envisaged as well.

Hitherto conventional methods for eliciting the immune response,immunization or vaccination are based on the use of DNA molecules inorder to incorporate the required genetic information into the cell.Various methods have been developed for introducing DNA into cells, suchas calcium phosphate transfection, polyprene transfection, protoplastfusion, electroporation, microinjection and lipofection, lipofectionhaving in particular proven to be a suitable method. DNA viruses maylikewise be used as a DNA vehicle. Because of their infectiousproperties, such viruses achieve a very high transfection rate. Theviruses used are genetically modified in such a manner that nofunctional infectious particles are formed in the transfected cell.Despite these precautions, however, it is not possible to rule out therisk of uncontrolled propagation of the introduced gene and viral genes,for example due to potential recombination events. This also entails therisk of the DNA being inserted into an intact gene of the host cell'sgenome by e.g. recombination, with the consequence that this gene may bemutated and thus completely or partially inactivated or may give rise tomisinformation. In other words, synthesis of a gene product which isvital to the cell may be completely suppressed or alternatively amodified or incorrect gene product is expressed. One particular riskoccurs if the DNA is integrated into a gene which is involved in theregulation of cell growth. In this case, the host cell may becomedegenerate and lead to cancer or tumor formation. Furthermore, if theDNA introduced into the cell is to be expressed, it is necessary for thecorresponding DNA vehicle to contain a strong promoter, such as theviral CMV promoter. The integration of such promoters into the genome ofthe treated cell may result in unwanted alterations of the regulation ofgene expression in the cell. Another risk of using DNA as an agent toinduce an immune response (e.g. as a vaccine) is the induction ofpathogenic anti-DNA antibodies in the patient into whom the foreign DNAhas been introduced, so bringing about a (possibly fatal) immuneresponse.

Thus overall, there is room and a need for an efficient system, whichmay be used to effectively stimulate the immune system to allowtreatment of lung cancer, especially of non-small cell lung cancer(NSCLC), while avoiding the problems of uncontrolled propagation of anintroduced gene due to DNA based compositions.

It is thus an object of the present invention to provide a composition,which a) allows treatment of lung cancer by stimulating the immunesystem, while b) avoiding the above mentioned disadvantages.

This object is solved by the subject matter of the present invention,particularly by an active (immunostimulatory) composition comprising atleast one RNA, encoding at least two (preferably different) antigensselected from the group comprising the antigens:

-   -   hTERT,    -   WT1,    -   MAGE-A2,    -   5T4,    -   MAGE-A3,    -   MUC1,    -   Her-2/neu,    -   NY-ESO-1,    -   CEA,    -   Survivin,    -   MAGE-C1, and/or    -   MAGE-C2.

Surprisingly, it has been found that a specific combination of at leasttwo antigens, antigenic proteins or antigenic peptides of the aforementioned group, as contained in an active (immunostimulatory)composition according to the present invention, is capable toeffectively stimulate the (adaptive) immune system to allow treatment oflung cancer, especially of non-small cell lung cancer (NSCLC). Herein,the terms antigens, antigenic proteins or antigenic peptides may be usedsynomously. In the context of the present invention, an active(immunostimulatory) composition according to the present invention shallbe further understood as a composition, which is able to elicit animmune response, preferably an adaptive immune response as definedherein, due to one of the component(s) contained or encoded by thecomponents of the active (immunostimulatory) composition, preferably bythe at least one RNA, preferably (m)RNA, encoding the at least two(preferably different) antigens.

The at least one RNA of the active (immunostimulatory) composition mayencode hTERT. In the context of this invention “hTERT” is humantelomerase reverse transcriptase and the preferred sequence of the RNA,preferably of the mRNA, encoding “hTERT”—if being used in the active(immunostimulatory) composition according to the invention—is shown inFIG. 7 (SEQ ID NO: 7), and—even more preferably, in FIG. 8 (SEQ ID NO:8). Minev, Hipp et al. (2000) described that telomerase is aribonucleoprotein enzyme which has been linked to malignanttransformation in human cells (Minev, B., J. Hipp, et al. (2000).“Cytotoxic T cell immunity against telomerase reverse transcriptase inhumans.” Proc Natl Acad Sci USA 97(9): 4796-801). Telomerase activity isincreased in the vast majority of human tumors, making its gene productthe first molecule common to all human tumors. The generation ofendogenously processed telomerase peptides bound to Class I MHCmolecules could therefore target cytotoxic T lymphocytes (CTL) to tumorsof different origins. Thus, according to them this could advance vaccinetherapy against cancer provided that precursor CTL recognizingtelomerase peptides in normal adults and cancer patients can be expandedthrough immunization. They further demonstrated that the majority ofnormal individuals and patients with prostate cancer immunized in vitroagainst two HLA-A2.1 restricted peptides from telomerase reversetranscriptase (hTRT) developed hTRT-specific CTL. Carpenter andVonderheide (2006) (Carpenter, E. L. and R. H. Vonderheide (2006);

“Telomerase-based immunotherapy of cancer.” Expert Opin Biol Ther 6(10):1031-9) reported that the progression from the cloning of humantelomerase reverse transcriptase (hTERT) in 1997 to the first clinicaltrials of hTERT as an antitumor immunotherapy target has been swift.hTERT is overexpressed in the vast majority of human cancers whereas ithas limited expression in normal adult tissue. It plays a critical rolein oncogenesis and may be expressed by cancer stem cells. However,despite being a self antigen, hTERT is immunogenic both in vitro and invivo. Several Phase I studies of hTERT immunotherapy have been completedin patients with breast, prostate, lung and other cancers, and clinicaland immunological results are encouraging. Immunotherapy inducedfunctional, antitumor T cells in patients in the absence of clinicaltoxicity. The opportunity for vaccinating individuals as animmunoprevention strategy can also be envisioned for hTERT-basedtherapies. Nair, S. K. and Heiser et al. (2000) described the inductionof anti-murine TERT immunity in mice vaccinated with dendritic cellstransduced with murine TERT RNA (see Nair, S. K., A. Heiser, et al.(2000). “Induction of cytotoxic T cell responses and tumor immunityagainst unrelated tumors using telomerase reverse transcriptase RNAtransfected dendritic cells.” Nat Med 6(9): 1011-7.). According to apreferred embodiment, the at least one RNA of the active(immunostimulatory) composition may thus encode an hTERT antigenselected from the sequence as shown in FIG. 7 (SEQ ID NO: 7), and—morepreferably, in FIG. 8 (SEQ ID NO: 8). According to a further preferredembodiment, the at least one RNA of the active (immunostimulatory)composition may alternatively or additionally encode an hTERT antigenselected from a fragment, a variant or an epitope of an hTERT sequenceas shown in FIG. 7 (SEQ ID NO: 7), and—more preferably, as shown in FIG.8 (SEQ ID NO: 8).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode WT1. In the context of this invention “WT1” is Wilmstumor 1 and the preferred sequence of the RNA, preferably of the mRNA,encoding “WT1”—if being used in the active (immunostimulatory)composition according to the invention—is shown in FIG. 9 (SEQ ID NO:9), more preferably in FIG. 10 (SEQ ID NO: 10), and—even morepreferably—in FIG. 11 (SEQ ID NO: 11). Oka, Y. A. and Tsuboi et al.(2004) found that Wilm's tumor protein is overexpressed in lung cancer(see Oka, Y., A. Tsuboi, et al. (2004). “Induction of WT1 (Wilms' tumorgene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and theresultant cancer regression.” Proc Natl Acad Sci USA 101(38): 13885-90).Oka et al. (2004, supra) vaccinated 10 patients with lung cancer with apeptide derived from WT1. They could show that clinical responsecorrelated with anti-tumor CD8+T cell activity. The Wilms' tumor geneWT1 is overexpressed in leukemias and various types of solid tumors, andthe WT1 protein was demonstrated to be an attractive target antigen forimmunotherapy against these malignancies. Oka et al. (2004, supra)reported the outcome of a phase I clinical study of WT1 peptide-basedimmunotherapy for patients with breast or lung cancer, myelodysplasticsyndrome, or acute myeloid leukemia. Twelve of the 20 patients for whomthe efficacy of WT1 vaccination could be assessed showed clinicalresponses such as reduction in leukemic blast cells or tumor sizesand/or tumor markers. A clear correlation was observed between anincrease in the frequencies of WT1-specific cytotoxic T lymphocytesafter WT1 vaccination and clinical responses. It was thereforedemonstrated that WT1 vaccination could induce WT1-specific cytotoxic Tlymphocytes and resulted in cancer regression without damage to normaltissues. According to a preferred embodiment, the at least one RNA ofthe active (immunostimulatory) composition may thus encode an WT1antigen selected from the sequence as shown in FIG. 9 (SEQ ID NO: 9),and—more preferably, as shown in FIG. 10 (SEQ ID NO: 10) and even morepreferably as shown in FIG. 11 (SEQ ID NO: 11). According to a furtherpreferred embodiment, the at least one RNA of the active(immunostimulatory) composition may alternatively or additionally encodean WT1 antigen selected from a fragment, a variant or an epitope of anWT1 sequence as shown in FIG. 9 (SEQ ID NO: 9), and—more preferably, asshown in FIG. 10 (SEQ ID NO: 10) and even more preferably as shown inFIG. 11 (SEQ ID NO: 11).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode MAGE-A2. In the context of this invention “MAGE-A2”is the melanoma antigen family A, 2B and the preferred sequence of theRNA, preferably of the mRNA, encoding “MAGE-A2”—if being used in theactive (immunostimulatory) composition according to the invention—isshown in FIG. 14 (SEQ ID NO: 14), and—even more preferably—in FIG. 15(SEQ ID NO: 15). Gillespie and Coleman (1999) (Gillespie, A. M. and R.E. Coleman (1999). “The potential of melanoma antigen expression incancer therapy.” Cancer Treat Rev 25(4): 219-27) reported expression inbladder cancer, breast cancer, colorectal cancer, gastric cancer, headand neck cancer, lung cancer, maxillary cancer, melanoma, oesophaguscancer, osteosarcoma and ovary cancer. According to a preferredembodiment, the at least one RNA of the active (immunostimulatory)composition may thus encode an MAGE-A2 antigen selected from thesequence as shown in FIG. 14 (SEQ ID NO: 14), and—more preferably, asshown in FIG. 15 (SEQ ID NO: 15). According to a further preferredembodiment, the at least one RNA of the active (immunostimulatory)composition may alternatively or additionally encode an MAGE-A2 antigenselected from a fragment, a variant or an epitope of an MAGE-A2 sequenceas shown in FIG. 14 (SEQ ID NO: 14), and—more preferably, as shown inFIG. 15 (SEQ ID NO: 15).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode 5T4. In the context of this invention “5T4” istrophoblast glycoprotein and the preferred sequence of the RNA,preferably of the mRNA, encoding “5T4”—if being used in the active(immunostimulatory) composition according to the invention—is shown inFIG. 3 (SEQ ID NO: 3), and—even more preferably—in FIG. 4 (SEQ ID NO:4). Harrop, Connolly et al. (2006) reported that the human oncofetalantigen 5T4 is a 72-kDa leucine-rich membrane glycoprotein which isexpressed at high levels on the placenta and also on a wide range ofhuman carcinomas including colorectal, gastric, renal, and ovariancancers but rarely on normal tissues (see Harrop, R., N. Connolly, etal. (2006). “Vaccination of colorectal cancer patients with modifiedVaccinia Ankara delivering the tumor antigen 5T4 (TroVax) induces immuneresponses which correlate with disease control: a phase I/II trial.”Clin Cancer Res 12(11 Pt 1): 3416-24). Overexpression of 5T4 isassociated with poor prognosis in patients with colorectal, gastric, andovarian carcinoma. Despite such compounding factors, 5T4-specificcellular and/or humoral immune responses were induced in the majority ofpatients (16 of 17; 94%) following TroVax immunization, which wasconsidered encouraging compared with many other cancer immunotherapytrials. In summary, they showed safety and immunogenicity of TroVaxdelivered via i.m. and i.d. routes of administration. Zhao and Wang(2007) (Zhao, Y. and Y. Wang (2007). “5T4 oncotrophoblast glycoprotein:janus molecule in life and a novel potential target against tumors.”Cell Mol Immunol 4(2): 99-104) reported that 5T4 oncotrophoblastglycoprotein is a transmembrane protein expressed on the embryonictissue and various malignant tumor cell surfaces. It plays a vital rolein the multiple biological and pathological processes including massivecellular migration during the embryogenesis, cell invasion associatedwith implantation, and neoplastic metastasis in the progression oftumorigenesis. According to Kopreski, Benko et al. (2001) 5T4 is atrophoblast glycoprotein frequently overexpressed in epithelialmalignancies that provides a potential target for cancer therapeutics(see Kopreski, M. S., F. A. Benko, et al. (2001). “Circulating RNA as atumor marker: detection of 5T4 mRNA in breast and lung cancer patientserum.” Ann N Y Acad Sci 945: 172-8). Serum was collected from 19patients with advanced breast cancer (5 patients) or non-small-cell lungcancer (14 patients), and from 25 normal control volunteers havingamplifiable RNA. RNA extracted from the serum was RT-PCR amplified usingheminested, two-stage reactions, with products detected by gelelectrophoresis. 5T4 mRNA was reproducibly detected in 8/19 (42%) cancerpatient sera, including 2/5 breast cancer patient sera and 6/14 lungcancer patient sera, but in only 3/25 (12%) normal control sera(p=0.035). According to a preferred embodiment, the at least one RNA ofthe active (immunostimulatory) composition may thus encode an 5T4antigen selected from the sequence as shown in FIG. 3 (SEQ ID NO: 3),and—more preferably, as shown in FIG. 4 (SEQ ID NO: 4). According to afurther preferred embodiment, the at least one RNA of the active(immunostimulatory) composition may alternatively or additionally encodean 5T4 antigen selected from a fragment, a variant or an epitope of an5T4 sequence as shown in FIG. 3 (SEQ ID NO: 3), and—more preferably, asshown in FIG. 4 (SEQ ID NO: 4).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode MAGE-A3. In the context of this invention “MAGE-A3”is the melanoma antigen family A, 3 and the preferred sequence of theRNA, preferably of the mRNA, encoding “MAGE-A3”—if being used in theactive (immunostimulatory) composition according to the invention—isshown in FIG. 16 (SEQ ID NO: 16), and—even more preferably—in FIG. 17(SEQ ID NO: 17). Gillespie and Coleman (1999) (Gillespie, A. M. and R.E. Coleman (1999). “The potential of melanoma antigen expression incancer therapy.” Cancer Treat Rev 25(4): 219-27) reported expression inbladder cancer, breast cancer, colorectal cancer, gastric cancer,glioma, head and neck cancer, lung, maxillary cancer, melanoma,neuroblastoma, oesophagus cancer and ovary cancer. Sienel, Varwark etal. (2004) described a study performed to determine the rate of MAGE-A3expression in early-stage non-small cell lung cancer (NSCLC) (seeSienel, W., C. Varwerk, et al. (2004). “Melanoma associated antigen(MAGE)-A3 expression in Stages I and II non-small cell lung cancer:results of a multi-center study.” Eur J Cardiothorac Surg 25(1): 131-4).Primary tumor samples from 204 patients with operable clinical stages Ior II NSCLC were collected and the pathological stage determined.MAGE-A3 expression was analyzed from tissue samples by detection ofMAGE-A3 transcripts using reverse-transcriptase polymerase chainreaction. MAGE-A3 expression was observed in 80 out of the 204 (39.2%)examined stages I-II primary tumors. Atanackovic, Altorki et al. (2004)described that MAGE-A3 a tumor-associated antigen originally identifiedin melanoma, was also found in non-small cell lung tumors (seeAtanackovic, D., N. K. Altorki, et al. (2004). “Vaccine-induced CD4+ Tcell responses to MAGE-3 protein in lung cancer patients.” J Immunol172(5): 3289-96). In a clinical trial nine NSCLC patients werevaccinated with the protein; 3 developed antibody responses. Seven of 8patients who received MAGE-A3 combined with adjuvant ASO2B generatedantibodies against MAGE-A3. Several of these patients also developed Tcell responses to the protein. According to a preferred embodiment, theat least one RNA of the active (immunostimulatory) composition may thusencode an MAGE-A3 antigen selected from the sequence as shown in FIG. 16(SEQ ID NO: 16), and—more preferably, as shown in FIG. 17 (SEQ ID NO:17). According to a further preferred embodiment, the at least one RNAof the active (immunostimulatory) composition may alternatively oradditionally encode an MAGE-A3 antigen selected from a fragment, avariant or an epitope of an MAGE-A3 sequence as shown in FIG. 16 (SEQ IDNO: 16), and—more preferably, as shown in FIG. 17 (SEQ ID NO: 17).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode MUC1. In the context of this invention “MUC1” ismucin 1 and the preferred sequence of the RNA, preferably of the mRNA,encoding “MUC1”—if being used in the active (immunostimulatory)composition according to the invention—is shown in FIG. 1 (SEQ ID NO:1), and—even more preferably—in FIG. 2 (SEQ ID NO: 2). Cancer-associatedmucins are a potential target for immunotherapy. These molecules arethought to promote metastases by facilitating adhesion of malignantcells to the endothelial cell surface. According to Denda-Nagai andIrimura (2000) (Denda-Nagai, K. and T. Irimura (2000). “MUC1 incarcinoma-host interactions.” Glycoconj J 17(7-9): 649-58) MUC-1 isoverexpressed in 90% of all adenocarcinomas, including breast, lung,pancreas, prostate, stomach, colon and ovary. Kontani, Taguchi et al.(2001) found that MUC-1 has been found to be expressed in 60% of lungcancers (see Kontani, K., O. Taguchi, et al. (2001). “Modulation of MUC1mucin as an escape mechanism of breast cancer cells from autologouscytotoxic T-lymphocytes.” Br J Cancer 84(9): 1258-64), whereas Kontani,Taguchi et al. (2003) found in a study analyzing the use of pulsed DCswith MUC1 antigens to elicit cellular immunity in MUC1 positive cancers,that clinically seven of nine MUC-1 positive patients responded to thetreatment with either a reduction in tumor marker levels ordisappearance of malignant pleural effusion (see Kontani, K., O.Taguchi, et al. (2003). “Dendritic cell vaccine immunotherapy of cancertargeting MUC1 mucin.” Int J Mol Med 12(4): 493-502). Three of theseresponding patients had NSCLC. Palmer, Parker et al. (2001) reportedthat in a phase I clinical trial using MUC1 peptide in stage III/IVNSCLC, safety and tolerability of this agent was established (seePalmer, M., J. Parker, et al. (2001). “Phase I study of the BLP25 (MUC1peptide) liposomal vaccine for active specific immunotherapy in stageIIIB/IV non-small-cell lung cancer.” Clin Lung Cancer 3(1): 49-57;discussion 58). Five of 12 patients (42%) had immunologic responses, and4 of 12 patients (33%) achieved stable disease. Wierecky, Mueller et al.(2006) further identified two HLA-A2 binding novel 9-mer peptides of theTAA MUC1, which is overexpressed on various hematological and epithelialmalignancies (see Wierecky, J., M. Mueller, et al. (2006). “Dendriticcell-based cancer immunotherapy targeting MUC-1.” Cancer ImmunolImmunother 55(1): 63-7). Cytotoxic T cells generated after pulsing DCwith these peptides were able to induce lysis of tumor cells expressingMUC1 in an antigen-specific and HLA-restricted fashion. Within twoclinical studies, it was demonstrated that vaccination of patients withadvanced cancer using DCs pulsed with MUC1 derived peptides was welltolerated without serious side effects and was able to induceimmunological responses. Of 20 patients with metastatic renal cellcarcinoma, 6 patients showed regression of metastases with 3 objectiveresponses (1 CR, 2 PR). According to a preferred embodiment, the atleast one RNA of the active (immunostimulatory) composition may thusencode an MUC1 antigen selected from the sequence as shown in FIG. 1(SEQ ID NO: 1), and—more preferably, as shown in FIG. 2 (SEQ ID NO: 2).According to a further preferred embodiment, the at least one RNA of theactive (immunostimulatory) composition may alternatively or additionallyencode an MUC1 antigen selected from a fragment, a variant or an epitopeof an MUC1 sequence as shown in FIG. 1 (SEQ ID NO: 1), and—morepreferably, as shown in FIG. 2 (SEQ ID NO: 2).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode Her-2/neu. In the context of this invention“Her-2/neu” is v-erb-b2 erythroblastic leukemia viral oncogene homolog 2and the preferred sequence of the RNA, preferably of the mRNA, encoding“Her-2/neu”—if being used in the active (immunostimulatory) compositionaccording to the invention—is shown in FIG. 5 (SEQ ID NO: 5), and—evenmore preferably—in FIG. 6 (SEQ ID NO: 6). According to Baxevanis,Sotiropolou et al. (2004) HER-2/neu (also known as HER2 or c-erb-B2) isa 185-kDa protein receptor with tyrosine kinase activity and extensivehomology to the epidermal growth factor (EGF) receptor (see Baxevanis,C. N., P. A. Sotiropoulou, et al. (2004). “Immunobiology of HER-2/neuoncoprotein and its potential application in cancer immunotherapy.”Cancer Immunol Immunother 53(3): 166-75). HER-2/neu is expressed in manyepithelial tumors and known to be overexpressed in approximately 20-25%of all ovarian and breast cancers, 35-45% of all pancreaticadenocarcinomas, and up to 90% of colorectal carcinomas. HER-2/neuoverexpression represents a marker of poor prognosis. Overexpression ofHer-2 has been observed in malignant tumors of the breast, ovary,pancreas, colon, lung and other tissues. Her-2 is normally expressed atlow levels in variety of human tissues (skin, digestive tractepithelium, breast, ovary, hepatocytes). Bernhard, Salazar (2002) reportin their conclusion that early results of clinical trials activelyimmunizing cancer patients against HER-2/neu demonstrated that immunitycould be generated and that immune responses persisted over a period oftime (see Bernhard, H., Salazar L., et al. (2002). “Vaccination againstthe HER-2/neu oncogenic protein.” Endocr Relat Cancer 9(1): 33-44).Current vaccine trials were focused solely on the use of epitope- orpeptide-based vaccines, largely due to the observation that peptidevaccine strategies could circumvent neu-specific tolerance in rodentmodels. The next generation of vaccine approaches according to Bernhardet al. (2002, supra) will likely include protein-based vaccines,HER-2/neu antigen preparations loaded onto DC, and nucleic acid basedformulations. Studies in rodent models exploring these strategies at apre-clinical level were promising. Expansion of HER-2/neu-specificT-cell ex vivo following active immunization or in vitro culture withHER-2/neu-expressing DC was thus considered as being a therapeuticoption for treating advanced stage HER-2/neu-overexpressing tumors.Baxevanis, Sotiridou et al. (2006) found that in humans, althoughimmunological responses have been detected against the peptides used forvaccination no clinical responses have been described (see Baxevanis, C.N., N. N. Sotiriadou, et al. (2006). “Immunogenic HER-2/neu peptides astumor vaccines.” Cancer Immunol Immunother 55(1): 85-95). According toDisis, Gooley et al. (2002) Her-2/neu is a member of the EGFR family(see Disis, M. L., T. A. Gooley, et al. (2002). “Generation of T-cellimmunity to the HER-2/neu protein after active immunization withHER-2/neu peptide-based vaccines.” J Clin Oncol 20(11): 2624-32). It isfrequently overexpressed in breast, ovary, prostate, colon and lungcancers. In a phase I clinical trial 38 patients (2 with NSCLC) werevaccinated with a Her-2/neu peptide. 92% of the patients developedT-cell immunity to Her-2/neu. According to a preferred embodiment, theat least one RNA of the active (immunostimulatory) composition may thusencode an Her-2/neu antigen selected from the sequence as shown in FIG.5 (SEQ ID NO: 5), and—more preferably, as shown in FIG. 6 (SEQ ID NO:6). According to a further preferred embodiment, the at least one RNA ofthe active (immunostimulatory) composition may alternatively oradditionally encode an Her-2/neu antigen selected from a fragment, avariant or an epitope of an Her-2/neu sequence as shown in FIG. 5 (SEQID NO: 5), and—more preferably, as shown in FIG. 6 (SEQ ID NO: 6).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode NY-ESO-1. In the context of this invention “NY-ESO-1”is cancer/testis antigen 1B and the preferred sequence of the RNA,preferably of the mRNA, encoding “NY-ESO-1”—if being used in the active(immunostimulatory) composition according to the invention—is shown inFIG. 20 (SEQ ID NO: 20), and—even more preferably—in FIG. 21 (SEQ ID NO:21). Chen, Scanlan et al. (1997) reported the mRNA expression ofNY-ESO-1 in various human tumors by RT-PCR finding Melanoma 23/67,Ovarian cancer 2/8, Breast cancer 10/33, Thyroid cancer 2/5, Prostatecancer 4/16, Bladder cancer 4/5, Colon cancer 0/16, Burkitt lymphoma1/2, Glioma 0/15, Basal cell carcinoma 0/2, Gastric cancer 0/12,Leiomyosarcoma 0/2, Lung cancer 2/12, Other sarcomas 0/2, Renal cancer0/10, Pancreatic cancer 0/2, Lymphoma 0/10, Seminoma 0/1, Hepatoma 2/7,Spinal cord tumor 0/1 (see Chen, Y. T., M. J. Scanlan, et al. (1997). “Atesticular antigen aberrantly expressed in human cancers detected byautologous antibody screening.” Proc Natl Acad Sci USA 94(5): 1914-8).Jager, Karbach et al. (2006) reported that NY-ESO-1 is a cancer/testisantigen expressed in a range of human malignancies, and that a number ofvaccine strategies targeting NY-ESO-1 were being developed (see Jager,E., J. Karbach, et al. (2006). “Recombinant vaccinia/fowlpox NY-ESO-1vaccines induce both humoral and cellular NY-ESO-1-specific immuneresponses in cancer patients.” Proc Natl Acad Sci USA 103(39): 14453-8).In the presented study, the safety and immunogenicity of recombinantvaccinia-NY-ESO-1 and recombinant fowlpox-NY-ESO-1 were analyzed in aseries of 36 patients with a range of different tumor types. Eachconstruct was first tested individually at two different dose levels andthen in a prime-boost setting with recombinant vaccinia-NY-ESO-1followed by recombinant fowlpox-NY-ESO-1. The vaccines were welltolerated either individually or together. NY-ESO-1-specific antibodyresponses and/or specific CD8 and CD4 T cell responses directed againsta broad range of NY-ESO-1 epitopes were induced by a course of at leastfour vaccinations at monthly intervals in a high proportion of patients.CD8 T cell clones derived from five vaccinated patients were shown tolyse NY-ESO-1-expressing melanoma target cells. In several patients withmelanoma, there was a strong impression that the natural course of thedisease was favorably influenced by vaccination. Davis, Chen et al.(2004) reported that HLA-A2-restricted NY-ESO-1 peptides injectedintradermally were shown to be safe and immunogenic (Davis, I. D., W.Chen, et al. (2004). “Recombinant NY-ESO-1 protein with ISCOMATRIXadjuvant induces broad integrated antibody and CD4(+) and CD8(+) T cellresponses in humans.” Proc Natl Acad Sci USA 101(29): 10697-702).Although these trials were designed only to determine safety andimmunogenicity, some patients showed tumor regression or stabilizationof disease. It was further expressed by Jager, Gnjatic et al. (2000)that a broad NY-ESO-1-specific immune response including antibody andCD4 and CD8 T cell responses was seen after immunization withrecombinant NY-ESO-1 protein combined with ISCOMATRIX adjuvant (CSLLtd., Parkville, Victoria, Australia) in patients with resectedNY-ESO-1-expressing melanoma (see Jager, E., S. Gnjatic, et al. (2000).“Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibodyresponses in peptide-vaccinated patients with NY-ESO-1+ cancers.” ProcNatl Acad Sci USA 97(22): 12198-203). This immune response to thevaccine appeared to be associated with long disease-free survival.Furthermore Odunsi, Qian et al. (2007) reported that vaccination with anNY-ESO-1 peptide induces integrated humoral and T cell responses inovarian cancer (see Odunsi, K., F. Qian, et al. (2007). “Vaccinationwith an NY-ESO-1 peptide of HLA class I/II specificities inducesintegrated humoral and T cell responses in ovarian cancer.” Proc NatlAcad Sci USA 104(31): 12837-42). According to a preferred embodiment,the at least one RNA of the active (immunostimulatory) composition maythus encode an NY-ESO-1 antigen selected from the sequence as shown inFIG. 20 (SEQ ID NO: 20), and—more preferably, as shown in FIG. 21 (SEQID NO: 21). According to a further preferred embodiment, the at leastone RNA of the active (immunostimulatory) composition may alternativelyor additionally encode an NY-ESO-1 antigen selected from a fragment, avariant or an epitope of an NY-ESO-1 sequence as shown in FIG. 20 (SEQID NO: 20), and—more preferably, as shown in FIG. 21 (SEQ ID NO: 21).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode CEA. In the context of this invention “CEA” iscarcinoembryonic antigen (CECAM5 =carcinoembryonic antigen-related celladhesion molecule 5) and the preferred sequence of the RNA, preferablyof the mRNA, encoding “CEA”—if being used in the active(immunostimulatory) composition according to the invention—is shown inFIG. 12 (SEQ ID NO: 12), and—even more preferably—in FIG. 13 (SEQ ID NO:13). According to Hammarstrom (1999) CEA is a 180 kDa onco-fetalglycoprotein that acts as an adhesion molecule, and is overexpressed in70% of NSCLC (Hammarstrom, S. (1999). “The carcinoembryonic antigen(CEA) family: structures, suggested functions and expression in normaland malignant tissues.” Semin Cancer Biol 9(2): 67-81). Berinstein(2002) reported that CEA has many attractive features as a target foractive vaccination approaches against cancer (Berinstein, N. L. (2002).“Carcinoembryonic antigen as a target for therapeutic anticancervaccines: a review.” J Clin Oncol 20(8): 2197-207). It has a favorableexpression pattern and is expressed in more than 50% of all humancancers. It may play a role in the tumorigenesis process itself, andthus its expression may be selected and conserved throughout cancerprogression. It has been well documented that CEA is processed andpresented on various MHC class 1 molecules. Moreover, immunologictolerance to CEA is not absolute. There are extensive data demonstratingthat human T cells can recognize, become activated to, and lyse cancercells that are expressing CEA. Several different therapeutic vaccinationapproaches using CEA as a target antigen have been assessed. The safetyof these approaches has been established. In addition, humoral and/orcellular responses to CEA have been documented. Although for the mostpart the patients chosen for these studies presented by Berinstein(2002, supra) had very advanced and refractory metastatic colon cancer,some evidence of clinical activity has been documented, with diseasestabilization and even objective responses occurring in some patients.Dendritic cells pulsed with an agonist CEA MHC class I binding peptide(CAP1-6D) and poxvirus-based vectors incorporating CEA, with or withoutcostimulatory molecules, seemed most active in activating CD8 T-cellresponses. Unfortunately, dendritic cell approaches may be limited bythe logistical difficulty of obtaining patient-specific preparations ofdendritic cells. Four phase I studies using the canarypox vector systemto target CEA were reported. These trials showed that such approacheswere safe, with mild grade 1 and grade 2 toxicities limited primarily tothe site of injection. Moreover, the trials showed that specificcellular T-cell responses can be activated to CEA in the majority ofpatients. These responses may be enhanced by the inclusion of the B7.1costimulatory molecule in the vector or by the addition of recombinantGM-CSF at the injection site. Although no objective clinical responseswere reported, a significant proportion of patients in these phase Istudies have experienced disease stabilization. Vaccination strategiesto further enhance the frequency of T cells recognizing CEA whereconsidered to further augment the clinical activity of these vaccines.There are data that suggest that at least some vaccines may be moreeffective in minimal disease states. Ueda, Itoh et al (2004) describedone study, in which 18 patients with metastatic gastrointestinal or lungcancer were treated with autologous dendritic cells pulsed withCEA-derived peptide (see Ueda, Y., T. Itoh, et al. (2004). “Dendriticcell-based immunotherapy of cancer with carcinoembryonicantigen-derived, HLA-A24-restricted CTL epitope: Clinical outcomes of 18patients with metastatic gastrointestinal or lung adenocarcinomas.” Int)Oncol 24(4): 909-17). Immune reactions measured by skin testing and invitro T cell assays were observed in most of the patients. Although noobjective clinical responses were reported, some patients had stabledisease while receiving this immunotherapy. According to a preferredembodiment, the at least one RNA of the active (immunostimulatory)composition may thus encode an CEA antigen selected from the sequence asshown in FIG. 12 (SEQ ID NO: 12), and—more preferably, as shown in FIG.13 (SEQ ID NO: 13). According to a further preferred embodiment, the atleast one RNA of the active (immunostimulatory) composition mayalternatively or additionally encode an CEA antigen selected from afragment, a variant or an epitope of an CEA sequence as shown in FIG. 12(SEQ ID NO: 12), and—more preferably, as shown in FIG. 13 (SEQ ID NO:13).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode Survivin. In the context of this invention “Survivin”is baculoviral IAP repeat-containing 5 (survivin) and the preferredsequence of the RNA, preferably of the mRNA, encoding “survivin”—ifbeing used in the active (immunostimulatory) composition according tothe invention—is shown in FIG. 18 (SEQ ID NO: 18), and—even morepreferably—in FIG. 19 (SEQ ID NO: 19). Grube, Moritz et al. (2007)described Survivin (see Grube, M., S. Moritz, et al. (2007). “CD8+Tcells reactive to survivin antigen in patients with multiple myeloma.”Clin Cancer Res 13(3): 1053-60). Survivin is a member of the inhibitorsof apoptosis family and is overexpressed in different types ofmalignancies. Cytotoxic T cells recognizing survivin epitopes can beelicited in vitro and by vaccination in patients with leukemia, breastcancer, and melanoma. It was investigated whether survivin-specific CD8+T cells occur in patients with multiple myeloma and T cells recognizingHLA-A2.1-binding survivin peptide were detected in 9 of 23 patients andin 1 of 21 healthy volunteers. Survivin-reactive T cells were identifiedas terminally differentiated effector T cells (CD8+, CD45RA+, andCCR7−). Positive survivin expression of myeloma cells in bone marrowspecimens was shown in 7 of 11 patients. Survivin is highly expressed inmost human cancer cells of epithelial and hematopoietic origin, andoverexpression is associated with cancer progression, poor prognosis,resistance, and short patient survival. Duffy, O'Donovan (2007)described that Survivin is a 16.5 kDa protein overexpressed in almostall malignancies but rarely detected in normal differentiated adulttissues (see Duffy, M. J., N. O'Donovan, et al. (2007). “Survivin: apromising tumor biomarker.” Cancer Lett 249(1): 49-60). Functionally,survivin has been shown to inhibit apoptosis, promote cell proliferationand enhance angiogenesis. Consistent with its role in these processes,survivin was described as playing a key role in cancer progression.Because of the large difference in expression between normal andmalignant tissue and its causal role in cancer progression, survivin iscurrently undergoing intensive investigation as a potential tumormarker. Emerging data suggests that measurement of survivin can aid theearly diagnosis of bladder cancer, determine prognosis in multiplecancer types and predict response to diverse anti-cancer therapies.Zeis, Siegel et al. (2003) demonstrated that human survivin-specificCTLs generated from PBMC by stimulation with autologous dendritic cellstransfected with survivin-RNA were cytotoxic for a range of hemopoieticmalignant cell lines and primary tumor cells isolated from patients withacute myeloid leukemia (see Zeis, M., S. Siegel, et al. (2003).“Generation of cytotoxic responses in mice and human individuals againsthematological malignancies using survivin-RNA-transfected dendriticcells.” J Immunol 170(11): 5391-7). It was also shown that vaccinationof mice with survivin-RNA-transfected dendritic cells lead to long termresistance to challenge by a survivin-expressing lymphoma, demonstratingthe potential of survivin as a tumor rejection Ag. Evidence for the useof survivin as a target structure for immunotherapeutic strategiesagainst hematological neoplasms was provided. According to a preferredembodiment, the at least one RNA of the active (immunostimulatory)composition may thus encode an Survivin antigen selected from thesequence as shown in FIG. 18 (SEQ ID NO: 18), and—more preferably, asshown in FIG. 19 (SEQ ID NO: 19). According to a further preferredembodiment, the at least one RNA of the active (immunostimulatory)composition may alternatively or additionally encode an Survivin antigenselected from a fragment, a variant or an epitope of an Survivinsequence as shown in FIG. 18 (SEQ ID NO: 18), and—more preferably, asshown in FIG. 19 (SEQ ID NO: 19).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode MAGE-C1. In the context of this invention “MAGE-C1”is the melanoma antigen family C, 1 and the preferred sequence of theRNA, preferably of the mRNA, encoding “MAGE-C1”—if being used in theactive (immunostimulatory) composition according to the invention—isshown in FIG. 22 (SEQ ID NO: 22), more preferably in FIG. 23 (SEQ ID NO:23), and—even more preferably—in FIG. 24 (SEQ ID NO: 24). Lucas, De Smetet al. (1998) recently identified MAGE-C1 by performing RDA (see Lucas,S., C. De Smet, et al. (1998). “Identification of a new MAGE gene withtumor-specific expression by representational difference analysis.”Cancer Res 58(4): 743-52). MAGE-C1 was not expressed in a panel ofnormal tissues tested with the exception of testis. Among tumoralsamples, MAGE-C1 was frequently expressed in seminomas, melanomas, andbladder carcinomas. It was also expressed in a significant fraction ofhead and neck carcinomas, breast carcinomas, non-small lung carcinomas,prostate adenocarcinomas and sarcomas. Jungbluth, Chen et al. (2002)described expression in breast cancer, ovary cancer, liver cancer,testis cancer, bladder cancer, melanoma and non-small cell lung cancer(39%) (see Jungbluth, A. A., Y. T. Chen, et al. (2002). “CT7 (MAGE-C1)antigen expression in normal and neoplastic tissues.” Int J Cancer99(6): 839-45). Gure, Chua et al. (2005) analyzed tumors from 523non-small-cell lung cancer (NSCLC) patients for the expression ofcancer-testis antigens (see Gure, A. O., R. Chua, et al. (2005).“Cancer-testis genes are coordinately expressed and are markers of pooroutcome in non-small cell lung cancer.” Clin Cancer Res 11(22):8055-62). MAGE-C1 was present in 18.8%. Scanlan, Altorki et al. (2000)furthermore reported expression of CT antigens in 33 non-small cell lungcancers: MAGE-C1: 30% (see Scanlan, M. J., N. K. Altorki, et al. (2000).“Expression of cancer-testis antigens in lung cancer: definition ofbromodomain testis-specific gene (BRDT) as a new CT gene, CT9.” CancerLett 150(2): 155-64). According to a preferred embodiment, the at leastone RNA of the active (immunostimulatory) composition may thus encode anMAGE-C1 antigen selected from the sequence as shown in FIG. 22 (SEQ IDNO: 22), and—more preferably, as shown in FIG. 23 (SEQ ID NO: 23) andeven more preferably as shown in FIG. 24 (SEQ ID NO: 24). According to afurther preferred embodiment, the at least one RNA of the active(immunostimulatory) composition may alternatively or additionally encodean MAGE-C1 antigen selected from a fragment, a variant or an epitope ofan MAGE-C1 sequence as shown in FIG. 22 (SEQ ID NO: 22), and—morepreferably, as shown in FIG. 23 (SEQ ID NO: 23) and even more preferablyas shown in FIG. 24 (SEQ ID NO: 24).

The at least one RNA of the active (immunostimulatory) composition mayfurthermore encode MAGE-C2. In the context of this invention “MAGE-C2”is the melanoma antigen family C2 and the preferred sequence of the RNA,preferably of the mRNA, encoding “MAGE-C2”—if being used in the active(immunostimulatory) composition according to the invention—is shown inFIG. 25 (SEQ ID NO: 25), and—even more preferably—in FIG. 26 (SEQ ID NO:26). Lucas, De Plaen et al. (2000) recently identified MAGE-C2 byperforming RDA on a melanoma cell line (see Lucas, S., E. De Plaen, etal. (2000). “MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3: four new members ofthe MAGE family with tumor-specific expression.” Int J Cancer 87(1):55-60). MAGE-C2 was not expressed in a panel of normal tissues testedwith the exception of testis. Among tumoral samples, MAGE-C2 wasfrequently expressed in seminomas, melanomas, and bladder carcinomas. Itwas also expressed in a significant fraction of head and neckcarcinomas, breast carcinomas, non-small lung carcinomas and sarcomas.Scanlan, Altorki et al. (2000) reported expression of CT antigens in 33non-small cell lung cancers: MAGE-C2: 30% (see Scanlan, M. J., N. K.Altorki, et al. (2000). “Expression of cancer-testis antigens in lungcancer: definition of bromodomain testis-specific gene (BRDT) as a newCT gene, CT9.” Cancer Lett 150(2): 155-64). According to a preferredembodiment, the at least one RNA of the active (immunostimulatory)composition may thus encode an MAGE-C2 antigen selected from thesequence as shown in FIG. 25 (SEQ ID NO: 25), and—more preferably, asshown in FIG. 26 (SEQ ID NO: 26). According to a further preferredembodiment, the at least one RNA of the active (immunostimulatory)composition may alternatively or additionally encode an MAGE-C2 antigenselected from a fragment, a variant or an epitope of an MAGE-C2 sequenceas shown in FIG. 25 (SEQ ID NO: 25), and—more preferably, as shown inFIG. 26 (SEQ ID NO: 26).

Antigens, antigenic proteins or antigenic peptides as defined abovewhich may be encoded by the at least one RNA of the active(immunostimulatory) composition according to the present invention, maycomprise fragments or variants of those sequences. Such fragments orvariants may typically comprise a sequence having a sequence homologywith one of the above mentioned antigens, antigenic proteins orantigenic peptides or sequences or their encoding nucleic acid sequencesof at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%,more preferably at least 80%, equally more preferably at least 85%, evenmore preferably at least 90% and most preferably at least 95% or even97%, to the entire wild-type sequence, either on nucleic acid level oron amino acid level.

“Fragments” of antigens, antigenic proteins or antigenic peptides in thecontext of the present invention may comprise a sequence of an antigen,antigenic protein or antigenic peptide as defined above, which is, withregard to its amino acid sequence (or its encoded nucleic acidsequence), N-terminally, C-terminally and/or intrasequentially truncatedcompared to the amino acid sequence of the original (native) protein (orits encoded nucleic acid sequence). Such truncation may thus occureither on the amino acid level or correspondingly on the nucleic acidlevel. A sequence homology with respect to such a fragment as definedabove may therefore preferably refer to the entire antigen, antigenicprotein or antigenic peptide as defined above or to the entire (coding)nucleic acid sequence of such an antigen, antigenic protein or antigenicpeptide.

Fragments of antigens, antigenic proteins or antigenic peptides in thecontext of the present invention may furthermore comprise a sequence ofan antigen, antigenic protein or antigenic peptide as defined above,which has a length of about 6 to about 20 or even more amino acids, e.g.fragments as processed and presented by MHC class I molecules,preferably having a length of about 8 to about 10 amino acids, e.g. 8,9, or 10, (or even 6, 7, 11, or 12 amino acids), or fragments asprocessed and presented by MHC class II molecules, preferably having alength of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19,20 or even more amino acids, wherein these fragments may be selectedfrom any part of the amino acid sequence. These fragments are typicallyrecognized by T-cells in form of a complex consisting of the peptidefragment and an MHC molecule, i.e. the fragments are typically notrecognized in their native form.

Fragments of antigens, antigenic proteins or antigenic peptides asdefined herein may also comprise epitopes of those antigens, antigenicproteins or antigenic peptides. Epitopes (also called “antigendeterminants”) in the context of the present invention are typicallyfragments located on the outer surface of (native) antigens, antigenicproteins or antigenic peptides as defined herein, preferably having 5 to15 amino acids, more preferably having 5 to 12 amino acids, even morepreferably having 6 to 9 amino acids, which may be recognized byantibodies or B-cell receptors, i.e. in their native form. Such epitopesof antigens, antigenic proteins or antigenic peptides may furthermore beselected from any of the herein mentioned variants of such antigens,antigenic proteins or antigenic peptides. In this context antigenicdeterminants can be conformational or discontinous epitopes which arecomposed of segments of the antigens, antigenic proteins or antigenicpeptides as defined herein that are discontinuous in the amino acidsequence of the antigens, antigenic proteins or antigenic peptides asdefined herein but are brought together in the three-dimensionalstructure or continuous or linear epitopes which are composed of asingle polypeptide chain.

“Variants” of antigens, antigenic proteins or antigenic peptides asdefined above may be encoded by the at least one RNA of the active(immunostimulatory) composition according to the present invention,wherein nucleic acids of the at least one (m)RNA, encoding the antigen,antigenic protein or antigenic peptide as defined above, are exchanged.Thereby, an antigen, antigenic protein or antigenic peptide may begenerated, having an amino acid sequence which differs from the originalsequence in one or more mutation(s), such as one or more substituted,inserted and/or deleted amino acid(s). Preferably, these fragmentsand/or variants have the same biological function or specific activitycompared to the full-length native antigen or antigenic protein, e.g.its specific antigenic property.

The at least one RNA of the active (immunostimulatory) compositionaccording to the present invention may also encode an antigen or anantigenic protein as defined above, wherein the encoded amino acidsequence comprises conservative amino acid substitution(s) compared toits physiological sequence. Those encoded amino acid sequences as wellas their encoding nucleotide sequences in particular fall under the termvariants as defined above. Substitutions in which amino acids whichoriginate from the same class are exchanged for one another are calledconservative substitutions. In particular, these are amino acids havingaliphatic side chains, positively or negatively charged side chains,aromatic groups in the side chains or amino acids, the side chains ofwhich can enter into hydrogen bridges, e.g. side chains which have ahydroxyl function.

This means that e.g. an amino acid having a polar side chain is replacedby another amino acid having a likewise polar side chain, or, forexample, an amino acid characterized by a hydrophobic side chain issubstituted by another amino acid having a likewise hydrophobic sidechain (e.g. serine (threonine) by threonine (serine) or leucine(isoleucine) by isoleucine (leucine)). Insertions and substitutions arepossible, in particular, at those sequence positions which cause nomodification to the three-dimensional structure or do not affect thebinding region. Modifications to a three-dimensional structure byinsertion(s) or deletion(s) can easily be determined e.g. using CDspectra (circular dichroism spectra) (Urry, 1985, Absorption, CircularDichroism and ORD of Polypeptides, in: Modern Physical Methods inBiochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam).

Furthermore, variants of antigens, antigenic proteins or antigenicpeptides as defined above, which may be encoded by the at least one RNAof the active (immunostimulatory) composition according to the presentinvention, may also comprise those sequences, wherein nucleic acids ofthe at least one (m)RNA are exchanged according to the degeneration ofthe genetic code, without leading to an alteration of respective aminoacid sequence of the antigen, antigenic protein or antigenic peptide,i.e. the amino acid sequence or at least part thereof may not differfrom the original sequence in one or more mutation(s) within the abovemeaning.

In order to determine the percentage to which two sequences (nucleicacid sequences, e.g. RNA or mRNA sequences as defined herein, or aminoacid sequences, preferably their encoded amino acid sequences, e.g. theamino acid sequences of the antigens, antigenic proteins or antigenicpeptides as defined above) are identical, the sequences can be alignedin order to be subsequently compared to one another. Therefore, e.g.gaps can be inserted into the sequence of the first sequence and thecomponent at the corresponding position of the second sequence can becompared. If a position in the first sequence is occupied by the samecomponent as is the case at a position in the second sequence, the twosequences are identical at this position. The percentage to which twosequences are identical is a function of the number of identicalpositions divided by the total number of positions. The percentage towhich two sequences are identical can be determined using a mathematicalalgorithm. A preferred, but not limiting, example of a mathematicalalgorithm which can be used is the algorithm of Karlin et al. (1993),PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res.,25:3389-3402. Such an algorithm is integrated in the BLAST program.Sequences which are identical to the sequences of the present inventionto a certain extent can be identified by this program.

The active (immunostimulatory) composition according to the presentinvention comprises, as defined above, at least one RNA, encoding leasttwo (preferably different) antigens selected from any of the antigens ofthe above group, since according to the invention a specific combinationof at least two (preferably different) antigens of the afore mentionedgroup is capable to effectively stimulate the (adaptive) immune systemto allow treatment of lung cancer, especially of non-small cell lungcancer (NSCLC). However, the present invention may also provide suchactive (immunostimulatory) compositions, comprising at least one RNA,encoding three, four, five, six, seven, eight, nine, ten, eleven or eveneven twelve (preferably different) antigens selected from any of theantigens of the above group, wherein any combination of these antigensis possible and envisaged.

According to a particularly preferred embodiment, the at least one RNAof the active (immunostimulatory) composition according to the presentinvention, may encode at least two (preferably different) antigensselected from any of the antigens of a subgroup comprising the followingantigens:

-   -   hTERT,    -   WT1,    -   5T4,    -   NY-ESO-1,    -   Survivin, and/or    -   MAGE-C2.

More preferably, the present invention may also provide an active(immunostimulatory) composition, comprising at least one RNA, encodingat least three, four, five or six (preferably different) antigensselected from any of the antigens of the above group or subgroup,wherein any combination of these antigens is possible.

Accordingly, due to another particularly preferred embodiment, the atleast one RNA of the active (immunostimulatory) composition of thepresent invention, may encode at least two (preferably different)antigens selected from any of the antigens of the above mentionedgroup(s) or subgroup(s) comprising (at least) any one of the followingcombinations of antigens:

-   -   hTERT and WT1, or    -   hTERT and 5T4, or    -   hTERT and NY-ESO-1, or    -   hTERT and Survivin, or    -   hTERT and MAGE-C2, or    -   WT1 and 5T4, or    -   WT1 and NY-ESO-1, or    -   WT1 and Survivin, or    -   WT1 and MAGE-C2, or    -   5T4 and NY-ESO-1, or    -   5T4 and Survivin, or    -   5T4 and MAGE-C2, or    -   NY-ESO-1 and Survivin, or    -   NY-ESO-1 and MAGE-C2, or    -   Survivin and MAGE-C2,    -   or    -   hTERT, WT1 and 5T4, or    -   hTERT, WT1 and NY-ESO-1, or    -   hTERT, WT1 and Survivin, or    -   hTERT, WT1 and MAGE-C2, or    -   hTERT, 5T4, and NY-ESO-1, or    -   hTERT, 5T4, and Survivin, or    -   hTERT, 5T4, and MAGE-C2, or    -   hTERT, NY-ESO-1 and Survivin, or    -   hTERT, NY-ESO-1 and MAGE-C2, or    -   hTERT, Survivin and MAGE-C2, or    -   WT1, 5T4 and NY-ESO-1, or    -   WT1, 5T4 and Survivin, or    -   WT1, 5T4 and MAGE-C2, or    -   WT1, NY-ESO-1 and Survivin, or    -   WT1, NY-ESO-1 and MAGE-C2, or    -   WT1, Survivin and MAGE-C2, or    -   5T4, NY-ESO-1 and Survivin, or    -   5T4, NY-ESO-1 and MAGE-C2, or    -   5T4, Survivin and MAGE-C2, or    -   NY-ESO-1, Survivin, and MAGE-C2,    -   or    -   hTERT, WT1, 5T4 and NY-ESO-1, or    -   hTERT, WT1, 5T4 and Survivin, or    -   hTERT, WT1, 5T4 and MAGE-C2, or    -   hTERT, 5T4, NY-ESO-1 and Survivin, or    -   hTERT, 5T4, NY-ESO-1 and MAGE-C2, or    -   hTERT, NY-ESO-1, Survivin and MAGE-C2, or    -   WT1, 5T4, NY-ESO-1, and Survivin, or    -   WT1, 5T4, NY-ESO-1, and MAGE-C2, or    -   WT1, 5T4, Survivin, and MAGE-C2, or    -   5T4, NY-ESO-1, Survivin, and MAGE-C2,    -   or    -   hTERT, WT1, 5T4, NY-ESO-1 and Survivin, or    -   hTERT, WT1, 5T4, NY-ESO-1 and MAGE-C2, or    -   WT1, 5T4, NY-ESO-1, Survivin and MAGE-C2,    -   or    -   hTERT, WT1, 5T4, NY-ESO-1, Survivin, and MAGE-C2.

More preferably, the at least one RNA of the active (immunostimulatory)composition of the present invention, may encode at least two(preferably different) antigens exclusively selected from any of theantigens of the above mentioned group(s) or subgroup(s) comprising (atleast) any one of the following combinations of antigens:

-   -   hTERT and WT1, or    -   hTERT and 5T4, or    -   hTERT and NY-ESO-1, or    -   hTERT and Survivin, or    -   hTERT and MAGE-C2, or    -   WT1 and 5T4, or    -   WT1 and NY-ESO-1, or    -   WT1 and Survivin, or    -   WT1 and MAGE-C2, or    -   5T4 and NY-ESO-1, or    -   5T4 and Survivin, or    -   5T4 and MAGE-C2, or    -   NY-ESO-1 and Survivin, or    -   NY-ESO-1 and MAGE-C2, or    -   Survivin and MAGE-C2,    -   or    -   hTERT, WT1 and 5T4, or    -   hTERT, WT1 and NY-ESO-1, or    -   hTERT, WT1 and Survivin, or    -   hTERT, WT1 and MAGE-C2, or    -   hTERT, 5T4, and NY-ESO-1, or    -   hTERT, 5T4, and Survivin, or    -   hTERT, 5T4, and MAGE-C2, or    -   hTERT, NY-ESO-1 and Survivin, or    -   hTERT, NY-ESO-1 and MAGE-C2, or    -   hTERT, Survivin and MAGE-C2, or    -   WT1, 5T4 and NY-ESO-1, or    -   WT1, 5T4 and Survivin, or    -   WT1, 5T4 and MAGE-C2, or    -   WT1, NY-ESO-1 and Survivin, or    -   WT1, NY-ESO-1 and MAGE-C2, or    -   WT1, Survivin and MAGE-C2, or    -   5T4, NY-ESO-1 and Survivin, or    -   5T4, NY-ESO-1 and MAGE-C2, or    -   5T4, Survivin and MAGE-C2, or    -   NY-ESO-1, Survivin, and MAGE-C2,    -   or    -   hTERT, WT1, 5T4 and NY-ESO-1, or    -   hTERT, WT1, 5T4 and Survivin, or    -   hTERT, WT1, 5T4 and MAGE-C2, or    -   hTERT, 5T4, NY-ESO-1 and Survivin, or    -   hTERT, 5T4, NY-ESO-1 and MAGE-C2, or    -   hTERT, NY-ESO-1, Survivin and MAGE-C2, or    -   WT1, 5T4, NY-ESO-1, and Survivin, or    -   WT1, 5T4, NY-ESO-1, and MAGE-C2, or    -   WT1, 5T4, Survivin, and MAGE-C2, or    -   5T4, NY-ESO-1, Survivin, and MAGE-C2,    -   or    -   hTERT, WT1, 5T4, NY-ESO-1 and Survivin, or    -   hTERT, WT1, 5T4, NY-ESO-1 and MAGE-C2, or    -   WT1, 5T4, NY-ESO-1, Survivin and MAGE-C2,    -   or    -   hTERT, WT1, 5T4, NY-ESO-1, Survivin, and MAGE-C2.

According to a further preferred embodiment, the present inventionprovides an active (immunostimulatory) composition comprising at leastone RNA, encoding at least two (preferably different) antigens,

-   a) wherein at least one, preferably at least two, three, four, five    or even six, of these at least two antigens is (are) selected from:    -   5T4    -   NY-ESO-1,    -   MAGE-A2,    -   MAGE-A3,    -   MAGE-C1, and/or    -   MAGE-C2, and-   b) wherein the further antigen(s) is (are) selected from at least    one antigen as defined herein, preferably in any of the herein    mentioned combinations, groups or subgroups of antigens, e.g. the    further antigen(s) is (are) selected from, e.g.:    -   hTERT,    -   WT1,    -   MAGE-A2,    -   5T4,    -   MAGE-A3,    -   MUC1,    -   Her-2/neu,    -   NY-ESO-1,    -   CEA,    -   Survivin,    -   MAGE-C1, and/or    -   MAGE-C2.

According to a further preferred embodiment, the at least one antigen(s)according to a) is (are) selected from:

-   -   NY-ESO-1,    -   MAGE-C1, and/or    -   MAGE-C2.

According to another preferred embodiment, the at least one antigen(s)according to a) is (are) selected from:

-   -   MAGE-C1, and/or    -   MAGE-C2.

According to another preferred embodiment, the at least one antigen(s)according to b) is (are) selected from an antigen (antigens) as definedin one of the following combinations:

-   -   hTERT and WT1; or    -   hTERT and MAGE-A2; or    -   hTERT and 5T4; or    -   hTERT and MAGE-A3; or    -   hTERT and MUC1; or    -   hTERT and Her-2/neu; or    -   hTERT and NY-ESO-1; or    -   hTERT and CEA; or    -   hTERT and Survivin; or    -   hTERT and MAGE-C1; or    -   hTERT and MAGE-C2; or    -   WT1 and MAGE-A2; or    -   WT1 and 5T4; or    -   WT1 and MAGE-A3; or    -   WT1 and MUC1; or    -   WT1 and Her-2/neu; or    -   WT1 and NY-ESO-1; or    -   WT1 and CEA; or    -   WT1 and Survivin; or    -   WT1 and MAGE-C1; or    -   WT1 and MAGE-C2; or    -   MAGE-A2 and 5T4; or    -   MAGE-A2 and MAGE-A3; or    -   MAGE-A2 and MUC1; or    -   MAGE-A2 and Her-2/neu; or    -   MAGE-A2 and NY-ESO-1; or    -   MAGE-A2 and CEA; or    -   MAGE-A2 and Survivin; or    -   MAGE-A2 and MAGE-C1; or    -   MAGE-A2 and MAGE-C2; or    -   5T4 and MAGE-A3; or    -   5T4 and MUC1; or    -   5T4 and Her-2/neu; or    -   5T4 and NY-ESO-1; or    -   5T4 and CEA; or    -   5T4 and Survivin; or    -   5T4 and MAGE-C1; or    -   5T4 and MAGE-C2; or    -   MAGE-A3 and MUC1; or    -   MAGE-A3 and Her-2/neu; or    -   MAGE-A3 and NY-ESO-1; or    -   MAGE-A3 and CEA; or    -   MAGE-A3 and Survivin; or    -   MAGE-A3 and MAGE-C1    -   MAGE-A3 and MAGE-C2    -   MUC1 and Her-2/neu; or    -   MUC1 and NY-ESO-1; or    -   MUC1 and CEA; or    -   MUC1 and Survivin; or    -   MUC1 and MAGE-C1; or    -   MUC1 and MAGE-C2; or    -   HER-2/NEU and NY-ESO-1; or    -   HER-2/NEU and CEA; or    -   HER-2/NEU and Survivin; or    -   HER-2/NEU and MAGE-C1; or    -   HER-2/NEU and MAGE-C2; or    -   NY-ESO-1 and CEA; or    -   NY-ESO-1 and Survivin; or    -   NY-ESO-1 and MAGE-C1; or    -   NY-ESO-1 and MAGE-C2; or    -   CEA and Survivin; or    -   CEA and MAGE-C1; or    -   CEA and MAGE-C2; or    -   Survivin and MAGE-C1; or    -   Survivin and MAGE-C2; or    -   MAGE-C1 and MAGE-C2;    -   or    -   hTERT, WT1 and MAGE-A2; or    -   hTERT, WT1 and 5T4; or    -   hTERT, WT1 and MAGE-A3; or    -   hTERT, WT1 and MUC1; or    -   hTERT, WT1 and Her-2/neu; or    -   hTERT, WT1 and NY-ESO-1; or    -   hTERT, WT1 and CEA; or    -   hTERT, WT1 and Survivin; or    -   hTERT, WT1 and MAGE-C1; or    -   hTERT, WT1 and MAGE-C2; or    -   WT1, MAGE-A2 and 5T4; or    -   WT1, MAGE-A2 and MAGE-A3; or    -   WT1, MAGE-A2 and MUC1; or    -   WT1, MAGE-A2 and Her-2/neu; or    -   WT1, MAGE-A2 and NY-ESO-1; or    -   WT1, MAGE-A2 and CEA; or    -   WT1, MAGE-A2 and Survivin; or    -   WT1, MAGE-A2 and MAGE-C1; or    -   WT1, MAGE-A2 and MAGE-C2; or    -   MAGE-A2, 5T4 and MAGE-A3; or    -   MAGE-A2, 5T4 and MUC1; or    -   MAGE-A2, 5T4 and Her-2/neu; or    -   MAGE-A2, 5T4 and NY-ESO-1; or    -   MAGE-A2, 5T4 and CEA; or    -   MAGE-A2, 5T4 and Survivin; or    -   MAGE-A2, 5T4 and MAGE-C1; or    -   MAGE-A2, 5T4 and MAGE-C2; or    -   5T4, MAGE-A3 and MUC1; or    -   5T4, MAGE-A3 and Her-2/neu; or    -   5T4, MAGE-A3 and NY-ESO-1; or    -   5T4, MAGE-A3 and CEA; or    -   5T4, MAGE-A3 and Survivin; or    -   5T4, MAGE-A3 and MAGE-C1; or    -   5T4, MAGE-A3 and MAGE-C2; or    -   MAGE-A3, MUC1 and Her-2/neu; or    -   MAGE-A3, MUC1 and NY-ESO-1; or    -   MAGE-A3, MUC1 and CEA; or    -   MAGE-A3, MUC1 and Survivin; or    -   MAGE-A3, MUC1 and MAGE-C1; or    -   MAGE-A3, MUC1 and MAGE-C2; or    -   MUC1, Her-2/neu and NY-ESO-1; or    -   MUC1, Her-2/neu and CEA; or    -   MUC1, Her-2/neu and Survivin; or    -   MUC1, Her-2/neu and MAGE-C1; or    -   MUC1, Her-2/neu and MAGE-C2; or    -   HER-2/NEU, NY-ESO-1 and CEA; or    -   HER-2/NEU, NY-ESO-1 and Survivin; or    -   HER-2/NEU, NY-ESO-1 and MAGE-C1; or    -   HER-2/NEU, NY-ESO-1 and MAGE-C2; or    -   NY-ESO-1, CEA and Survivin; or    -   NY-ESO-1, CEA and MAGE-Ci; or    -   NY-ESO-1, CEA and MAGE-C2; or    -   CEA, Survivin and MAGE-C1; or    -   CEA, Survivin and MAGE-C2; or    -   Survivin, MAGE-C1 and MAGE-C2;    -   or    -   hTERT, WT1, MAGE-A2 and 5T4; or    -   hTERT, WT1, MAGE-A2 and MAGE-A3; or    -   hTERT, WT1, MAGE-A2 and MUC1; or    -   hTERT, WT1, MAGE-A2 and Her-2/neu; or    -   hTERT, WT1, MAGE-A2 and NY-ESO-1; or    -   hTERT, WT1, MAGE-A2 and CEA; or    -   hTERT, WT1, MAGE-A2 and Survivin; or    -   hTERT, WT1, MAGE-A2 and MAGE-C1; or    -   hTERT, WT1, MAGE-A2 and MAGE-C2; or    -   WTI, MAGE-A2, 5T4 and MAGE-A3; or    -   WT1, MAGE-A2, 5T4 and MUC1; or    -   WT1, MAGE-A2, 5T4 and Her-2/neu; or    -   WT1, MAGE-A2, 5T4 and NY-ESO-1; or    -   WT1, MAGE-A2, 5T4 and CEA; or    -   WT1, MAGE-A2, 5T4 and Survivin; or    -   WT1, MAGE-A2, 5T4 and MAGE-C1; or    -   WT1, MAGE-A2, 5T4 and MAGE-C2; or    -   MAGE-A2, 5T4, MAGE-A3 and MUC1; or    -   MAGE-A2, 5T4, MAGE-A3 and Her-2/neu; or    -   MAGE-A2, 5T4, MAGE-A3 and NY-ESO-1; or    -   MAGE-A2, 5T4, MAGE-A3 and CEA; or    -   MAGE-A2, 5T4, MAGE-A3 and Survivin; or    -   MAGE-A2, 5T4, MAGE-A3 and MAGE-C1; or    -   MAGE-A2, 5T4, MAGE-A3 and MAGE-C2; or    -   5T4, MAGE-A3, MUC1, and Her-2/neu; or    -   5T4, MAGE-A3, MUC1 and NY-ESO-1; or    -   5T4, MAGE-A3, MUC1 and CEA; or    -   5T4, MAGE-A3, MUC1 and Survivin; or    -   5T4, MAGE-A3, MUC1 and MAGE-C1; or    -   5T4, MAGE-A3, MUC1 and MAGE-C2; or    -   MAGE-A3, MUC1, Her-2/neu and NY-ESO-1; or    -   MAGE-A3, MUC1, Her-2/neu and CEA; or    -   MAGE-A3, MUC1, Her-2/neu and Survivin; or    -   MAGE-A3, MUC1, Her-2/neu and MAGE-C1; or    -   MAGE-A3, MUC1, Her-2/neu and MAGE-C2; or    -   MUC1, Her-2/neu, NY-ESO-1 and CEA; or    -   MUC1, Her-2/neu, NY-ESO-1 and Survivin; or    -   MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1; or    -   MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2; or    -   HER-2/NEU, NY-ESO-1, CEA and Survivin; or    -   HER-2/NEU, NY-ESO-1, CEA and MAGE-C1; or    -   HER-2/NEU, NY-ESO-1, CEA and MAGE-C2; or    -   NY-ESO-1, CEA, Survivin and MAGE-C1; or    -   NY-ESO-1, CEA, Survivin and MAGE-C2; or    -   CEA, Survivin, MAGE-C1 and MAGE-C2;    -   or    -   hTERT, WT1, MAGE-A2, 5T4 and MAGE-A3; or    -   hTERT, WT1, MAGE-A2, 5T4 and MUC1; or    -   hTERT, WT1, MAGE-A2, 5T4 and Her-2/neu; or    -   hTERT, WT1, MAGE-A2, 5T4 and NY-ESO-1; or    -   hTERT, WT1, MAGE-A2, 5T4 and CEA; or    -   hTERT, WT1, MAGE-A2, 5T4 and Survivin; or    -   hTERT, WT1, MAGE-A2, 5T4 and MAGE-C1; or    -   hTERT, WT1, MAGE-A2, 5T4 and MAGE-C2; or    -   WT1, MAGE-A2, 5T4, MAGE-A3 and MUC1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3 and Her-2/neu; or    -   WT1, MAGE-A2, 5T4, MAGE-A3 and NY-ESO-1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3 and CEA; or    -   WT1, MAGE-A2, 5T4, MAGE-A3 and Survivin; or    -   WT1, MAGE-A2, 5T4, MAGE-A3 and MAGE-C1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3 and MAGE-C2; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1 and Her-2/neu; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1 and NY-ESO-1; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1 and Survivin; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C1; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C2; or    -   5T4, MAGE-A3, MUC1, Her-2/neu and NY-ESO-1; or    -   5T4, MAGE-A3, MUC1, Her-2/neu and CEA; or    -   5T4, MAGE-A3, MUC1, Her-2/neu and Survivin; or    -   5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C1; or    -   5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C2; or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and CEA; or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and Survivin; or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1; or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2; or    -   MUC1, Her-2/neu, NY-ESO-1, CEA and Survivin; or    -   MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1; or    -   MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2; or    -   HER-2/NEU, NY-ESO-1, CEA, Survivin and MAGE-C1; or    -   HER-2/NEU, NY-ESO-1, CEA, Survivin and MAGE-C2; or    -   NY-ESO-1, CEA, Survivin, MAGE-C1 and MAGE-C2;    -   or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and MUC1; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and Her-2/neu; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and NY-ESO-1; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and CEA; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and Survivin; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and MAGE-C1; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and MAGE-C2; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and Her-2/neu; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and NY-ESO-1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and Survivin; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C2; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and NY-ESO-1; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and CEA; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and Survivin; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C1; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C2; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and CEA; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and Survivin; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2; or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and Survivin; or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1; or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2; or    -   MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C1; or    -   MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C2; or    -   HER-2/NEU, NY-ESO-1, CEA, Survivin, MAGE-C1 and MAGE-C2;    -   or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and Her-2/neu; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and NY-ESO-1; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and Survivin; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C1; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C2; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and NY-ESO-1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and CEA; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and Survivin; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C2; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and CEA; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and Survivin;        or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and Survivin; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2; or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C1;        or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C2;        or    -   MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1 and MAGE-C2;    -   or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and NY-ESO-1;        or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and CEA; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and Survivin;        or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C1;        or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C2;        or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and CEA;        or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and        Survivin; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and        MAGE-C1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and        MAGE-C2; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and        Survivin; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and        MAGE-C1; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and        MAGE-C2; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and        MAGE-C1; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and        MAGE-C2; or    -   MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1 and        MAGE-C2;    -   or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and        CEA; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and        Survivin; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and        MAGE-C1; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and        MAGE-C2; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and        Survivin; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and        MAGE-C1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and        MAGE-C2; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin        and MAGE-C1; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin        and MAGE-C2; or    -   5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1        and MAGE-C2;    -   or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1,        CEA and Survivin; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1,        CEA and MAGE-C1; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1,        CEA and MAGE-C2; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA,        Survivin and MAGE-C1; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA,        Survivin and MAGE-C2; or    -   MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin,        MAGE-C1 and MAGE-C2;    -   or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1,        CEA, Survivin and MAGE-C1; or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1,        CEA, Survivin and MAGE-C2; or    -   WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA,        Survivin, MAGE-C1 and MAGE-C2;    -   or    -   hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1,        CEA, Survivin, MAGE-C1 and MAGE-C2.

According to another particularly preferred embodiment, the at least oneantigen(s) according to b) is (are) selected from the following specificcombination of antigens as defined above:

-   -   Survivin and 5T4

The at least one RNA of the active (immunostimulatory) compositionaccording to the present invention is typically any RNA, preferably,without being limited thereto, a coding RNA, a circular or linear RNA, asingle- or a double-stranded RNA (which may also be regarded as a RNAdue to non-covalent association of two single-stranded RNA) or apartially double-stranded or partially single stranded RNA, which are atleast partially self complementary (both of these partiallydouble-stranded or partially single stranded RNA molecules are typicallyformed by a longer and a shorter single-stranded RNA molecule or by twosingle stranded RNA-molecules, which are about equal in length, whereinone single-stranded RNA molecule is in part complementary to the othersingle-stranded RNA molecule and both thus form a double-stranded RNA inthis region, i.e. a partially double-stranded or partially singlestranded RNA with respect to the entire RNA sequence). More preferably,the at least one RNA of the active (immunostimulatory) compositionaccording to the present invention is a single-stranded RNA, even morepreferably a linear RNA. Most preferably, the at least RNA of the active(immunostimulatory) composition according to the present invention is amessenger RNA (mRNA). In this context, a messenger RNA (mRNA) istypically a RNA, which is composed of (at least) several structuralelements, e.g. an optional 5′-UTR region, an upstream positionedribosomal binding site followed by a coding region, an optional 3′-UTRregion, which may be followed by a poly-A tail (and/or a poly-C-tail).

Due to one particularly preferred embodiment, each of the at least two(preferably different) antigens of the active (immunostimulatory)composition of the present invention, may be encoded by one(monocistronic) RNA, preferably one (monocistronic) mRNA. In otherwords, the active (immunostimulatory) composition of the presentinvention may contain at least two (monocistronic) RNAs, preferablymRNAs, wherein each of these at least two (monocistronic) RNAs,preferably mRNAs, may encode just one (preferably different) antigen,selected from one of the above mentioned groups or subgroups, preferablyin one of the above mentioned combinations.

According to another particularly preferred embodiment, the active(immunostimulatory) composition of the present invention, may comprise(at least) one bi- or even multicistronic RNA, preferably mRNA, i.e. (atleast) one RNA which carries two or even more of the coding sequences ofat the least two (preferably different) antigens, selected from one ofthe above mentioned groups or subgroups, preferably in one of the abovementioned combinations. Such coding sequences of the at least two(preferably different) antigens of the (at least) one bi- or evenmulticistronic RNA may be separated by at least one IRES (internalribosomal entry site) sequence, as defined below. Thus, the term“encoding at least two (preferably different) antigens” may mean,without being limited thereto, that the (at least) one (bi- or evenmulticistronic) RNA, preferably a mRNA, may encode e.g. at least two,three, four, five, six, seven, eight, nine, ten, eleven or twelve(preferably different) antigens of the above mentioned group(s) ofantigens or their fragments or variants within the above definitions.More preferably, without being limited thereto, the (at least) one (bi-or even multicistronic) RNA, preferably mRNA, may encode e.g. at leasttwo, three, four, five or six (preferably different) antigens of theabove mentioned subgroup(s) of antigens or their fragments or variantswithin the above definitions. In this context, a so-called IRES(internal ribosomal entry site) sequence as defined above can functionas a sole ribosome binding site, but it can also serve to provide a bi-or even multicistronic RNA as defined above which codes for severalproteins, which are to be translated by the ribosomes independently ofone another. Examples of IRES sequences which can be used according tothe invention are those from picornaviruses (e.g. FMDV), pestiviruses(CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot andmouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swinefever viruses (CSFV), mouse leukoma virus (MLV), simian immunodeficiencyviruses (SIV) or cricket paralysis viruses (CrPV).

According to a further particularly preferred embodiment, the active(immunostimulatory) composition of the present invention, may comprise amixture of at least one monocistronic RNA, preferably mRNA, as definedabove, and at least one bi- or even multicistronic RNA, preferably mRNA,as defined above. The at least one monocistronic RNA and/or the at leastone bi- or even multicistronic RNA preferably encode different antigensor their fragments or variants within the above definitions, theantigens preferably being selected from one of the above mentionedgroups or subgroups of antigens, more preferably in one of the abovementioned combinations. However, the at least one monocistronic RNA andthe at least one bi- or even multicistronic RNA may preferably alsoencode (in part) identical antigens selected from one of the abovementioned groups or subgroups of antigens, preferably in one of theabove mentioned combinations, provided that the active(immunostimulatory) composition of the present invention as a wholeprovides at least two (preferably different) antigens as defined above.Such an embodiment may be advantageous e.g. for a staggered, e.g. timedependent, administration of the active (immunostimulatory) compositionof the present invention to a patient in need thereof. The components ofsuch an active (immunostimulatory) composition of the present invention,particularly the different RNAs encoding the at least two (preferablydifferent) antigens, may be e.g. contained in (different parts of) a kitof parts composition or may be e.g. administered separately ascomponents of different active (immunostimulatory) compositionsaccording to the present invention.

Preferably, the at least one RNA of the active (immunostimulatory)composition, encoding at least two (preferably different) antigensselected from the above defined group or subgroup of antigens, morepreferably in the above combinations, typically comprises a length ofabout 50 to about 20000, or 100 to about 20000 nucleotides, preferablyof about 250 to about 20000 nucleotides, more preferably of about 500 toabout 10000, even more preferably of about 500 to about 5000.

According to one embodiment, the at least one RNA of the active(immunostimulatory) composition, encoding at least two (preferablydifferent) antigens selected from the above defined group(s) orsubgroup(s) of antigens, more preferably in the above combinations, maybe in the form of a modified RNA, wherein any modification, as definedherein, may be introduced into the at least one RNA of the active(immunostimulatory) composition. Modifications as defined hereinpreferably lead to a stabilized at least one RNA of the active(immunostimulatory) composition of the present invention.

According to a first embodiment, the at least one RNA of the active(immunostimulatory) composition of the present invention may thus beprovided as a “stabilized RNA”, preferably a stabilized mRNA, that is tosay as an (m)RNA that is essentially resistant to in vivo degradation(e.g. by an exo- or endo-nuclease). Such stabilization can be effected,for example, by a modified phosphate backbone of the at least one (m)RNAof the active (immunostimulatory) composition of the present invention.A backbone modification in connection with the present invention is amodification in which phosphates of the backbone of the nucleotidescontained in the RNA are chemically modified. Nucleotides that may bepreferably used in this connection contain e.g. aphosphorothioate-modified phosphate backbone, preferably at least one ofthe phosphate oxygens contained in the phosphate backbone being replacedby a sulfur atom. Stabilized (m)RNAs may further include, for example:non-ionic phosphate analogues, such as, for example, alkyl and arylphosphonates, in which the charged phosphonate oxygen is replaced by analkyl or aryl group, or phosphodiesters and alkylphosphotriesters, inwhich the charged oxygen residue is present in alkylated form. Suchbackbone modifications typically include, without implying anylimitation, modifications from the group consisting ofmethylphosphonates, phosphoramidates and phosphorothioates (e.g.cytidine-5′-O-(1-thiophosphate)).

The at least one RNA of the active (immunostimulatory) composition ofthe present invention may additionally or alternatively also containsugar modifications. A sugar modification in connection with the presentinvention is a chemical modification of the sugar of the nucleotides ofthe at least one RNA and typically includes, without implying anylimitation, sugar modifications selected from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide(2′-fluoro-2′-deoxycytidine-5′-triphosphate,2′-fluoro-2′-deoxyuridine-5′-triphosphate), 2′-deoxy-2′-deamineoligoribonucleotide (2′-amino-2′-deoxycytidine-5′-triphosphate,2′-amino-2′-deoxyuridine-5′-triphosphate), 2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyl oligoribonucleotide(2′-O-methylcytidine-5′-triphosphate, 2′-methyluridine-5′-triphosphate), 2′-C-alkyl oligoribonucleotide, and isomers thereof(2′-aracytidine-5′-triphosphate, 2′-arauridine-5′-triphosphate), orazidotriphosphate (2′-azido-2′-deoxycytidine-5′-triphosphate,2′-azido-2′-deoxyuridine-5′-triphosphate).

The at least one RNA of the active (immunostimulatory) composition ofthe present invention may additionally or alternatively also contain atleast one base modification, which is preferably suitable for increasingthe expression of the protein coded for by the at least one RNA sequencesignificantly as compared with the unaltered, i.e. natural (=native),RNA sequence. Significant in this case means an increase in theexpression of the protein compared with the expression of the native RNAsequence by at least 20%, preferably at least 30%, 40%, 50% or 60%, morepreferably by at least 70%, 80%, 90% or even 100% and most preferably byat least 150%, 200% or even 300% or more. In connection with the presentinvention, a nucleotide having such a base modification is preferablyselected from the group of the base-modified nucleotides consisting of2-amino-6-chloropurineriboside-5′-triphosphate,2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate,5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate,6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

According to another embodiment, the at least one RNA of the active(immunostimulatory) composition of the present invention can likewise bemodified (and preferably stabilized) by introducing further modifiednucleotides containing modifications of their ribose or base moieties.Generally, the at least one (m)RNA of the active (immunostimulatory)composition of the present invention may contain any native (=naturallyoccurring) nucleotide, e.g. guanosine, uracil, adenosine, and/orcytosine or an analogue thereof. In this connection, nucleotideanalogues are defined as non-natively occurring variants of naturallyoccurring nucleotides. Accordingly, analogues are chemically derivatizednucleotides with non-natively occurring functional groups, which arepreferably added to or deleted from the naturally occurring nucleotideor which substitute the naturally occurring functional groups of anucleotide. Accordingly, each component of the naturally occurringnucleotide may be modified, namely the base component, the sugar(ribose) component and/or the phosphate component forming the backbone(see above) of the RNA sequence. Analogues of guanosine, uracil,adenosine, and cytosine include, without implying any limitation, anynaturally occurring or non-naturally occurring guanosine, uracil,adenosine, thymidine or cytosine that has been altered chemically, forexample by acetylation, methylation, hydroxylation, etc., including1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine,2,2-dimethyl-guanosine, 2,6-diaminopurine, 2′-Amino-2′-deoxyadenosine,2′-Amino-2′-deoxycytidine, 2′-Amino-2′-deoxyguanosine,2′-Amino-2′-deoxyuridine, 2-Amino-6-chloropurineriboside,2-Aminopurine-riboside, 2′-Araadenosine, 2′-Aracytidine, 2′-Arauridine,2′-Azido-2∝-deoxyadenosine, 2′-Azido-2′-deoxycytidine,2′-Azido-2′-deoxyguanosine, 2′-Azido-2′-deoxyuridine, 2-Chloroadenosine,2′-Fluoro-2′-deoxyadenosine, 2′-Fluoro-2′-deoxycytidine,2′-Fluoro-2′-deoxyguanosine, 2′-Fluoro-2′-deoxyuridine,2′-Fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine,2-methyl-thio-N6-isopenenyl-adenosine, 2′-O-Methyl-2-aminoadenosine,2′-O-Methyl-2′-deoxyadenosine, 2′-O-Methyl-2′-deoxycytidine,2′-O-Methyl-2′-deoxyguanosine, 2′-O-Methyl-2′-deoxyuridine,2′-O-Methyl-5-methyluridine, 2′-O-Methylinosine,2′-O-Methylpseudouridine, 2-Thiocytidine, 2-thio-cytosine,3-methyl-cytosine, 4-acetyl-cytosine, 4-Thiouridine,5-(carboxyhydroxymethyl)-uracil, 5,6-Dihydrouridine,5-Aminoallylcytidine, 5-Aminoallyl-deoxy-uridine, 5-Bromouridine,5-carboxymehtylaminomethyl-2-thio-uracil,5-carboxymethylamonomethyl-uracil, 5-Chloro-Ara-cytosine,5-Fluoro-uridine, 5-Iodouridine, 5-methoxycarbonylmethyl-uridine,5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-Azacytidine, 6-Azauridine,6-Chloro-7-deaza-guanosine, 6-Chloropurineriboside,6-Mercapto-guanosine, 6-Methyl-mercaptopurine-riboside,7-Deaza-2′-deoxy-guanosine, 7-Deazaadenosine, 7-methyl-guanosine,8-Azaadenosine, 8-Bromo-adenosine, 8-Bromo-guanosine,8-Mercapto-guanosine, 8-Oxoguanosine, Benzimidazole-riboside,Beta-D-mannosyl-queosine, Dihydro-uracil, Inosine, N1-Methyladenosine,N6-([6-Aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine,N6-methyl-adenosine, N7-Methyl-xanthosine, N-uracil-5-oxyacetic acidmethyl ester, Puromycin, Queosine, Uracil-5-oxyacetic acid,Uracil-5-oxyacetic acid methyl ester, Wybutoxosine, Xanthosine, andXylo-adenosine. The preparation of such analogues is known to a personskilled in the art, for example from U.S. Pat. No. 4,373,071, U.S. Pat.No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S.Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679,U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No.5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642. In the case of ananalogue as described above, particular preference may be givenaccording to the invention to those analogues that increase theimmunogenity of the RNA of the inventive active (immunostimulatory)composition and/or do not interfere with a further modification of theRNA that has been introduced.

According to a particular embodiment, the at least one RNA of the active(immunostimulatory) composition of the present invention can contain alipid modification. Such a lipid-modified RNA typically comprises a RNAas defined herein, encoding at least two antigens selected from thegroup or subgroup of antigens as defined above, preferably in the abovecombinations. Such a lipid-modified RNA typically further comprises atleast one linker covalently linked with that RNA, and at least one lipidcovalently linked with the respective linker. Alternatively, thelipid-modified RNA comprises an at least one RNA as defined herein andat least one (bifunctional) lipid covalently linked (without a linker)with that RNA. According to a third alternative, the lipid-modified RNAcomprises a RNA as defined herein, at least one linker covalently linkedwith that RNA, and at least one lipid covalently linked with therespective linker, and also at least one (bifunctional) lipid covalentlylinked (without a linker) with that RNA.

The lipid contained in the at least one RNA of the inventive active(immunostimulatory) composition (complexed or covalently bound thereto)is typically a lipid or a lipophilic residue that preferably is itselfbiologically active. Such lipids preferably include natural substancesor compounds such as, for example, vitamins, e.g. alpha-tocopherol(vitamin E), including RRR-alpha-tocopherol (formerlyD-alpha-tocopherol), L-alpha-tocopherol, the racemateD,L-alpha-tocopherol, vitamin E succinate (VES), or vitamin A and itsderivatives, e.g. retinoic acid, retinol, vitamin D and its derivatives,e.g. vitamin D and also the ergosterol precursors thereof, vitamin E andits derivatives, vitamin K and its derivatives, e.g. vitamin K andrelated quinone or phytol compounds, or steroids, such as bile acids,for example cholic acid, deoxycholic acid, dehydrocholic acid,cortisone, digoxygenin, testosterone, cholesterol or thiocholesterol.Further lipids or lipophilic residues within the scope of the presentinvention include, without implying any limitation, polyalkylene glycols(Oberhauser et at, Nucl. Acids Res., 1992, 20, 533), aliphatic groupssuch as, for example, C1-C20-alkanes, C1-C20-alkenes or C1-C20-alkanolcompounds, etc., such as, for example, dodecanediol, hexadecanol orundecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 111;Kabanov et al, FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie,1993, 75, 49), phospholipids such as, for example, phosphatidylglycerol,diacylphosphatidylglycerol, phosphatidylcholine,dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, di-hexadecyl-rac-glycerol,sphingolipids, cerebrosides, gangliosides, or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al,Tetrahedron Lett., 1995, 36, 3651; Shea et al, Nucl. Acids Res., 1990,18, 3777), polyamines or polyalkylene glycols, such as, for example,polyethylene glycol (PEG) (Manoharan et al, Nucleosides & Nucleotides,1995, 14, 969), hexaethylene glycol (HEG), palmitin or palmityl residues(Mishra et al, Biochim. Biophys. Acta, 1995, 1264, 229), octadecylaminesor hexylamino-carbonyl-oxycholesterol residues (Crooke et al, J.Pharmacol. Exp. Ther., 1996, 277, 923), and also waxes, terpenes,alicyclic hydrocarbons, saturated and mono- or poly-unsaturated fattyacid residues, etc.

The at least one RNA of the active (immunostimulatory) composition ofthe present invention may likewise be stabilized in order to preventdegradation of the RNA in vivo by various approaches. It is known in theart that instability and (fast) degradation of mRNA or of RNA in vivo ingeneral may represent a serious problem in the application of RNA basedcompositions. This instability of RNA is typically due to RNA-degradingenzymes, “RNAases” (ribonucleases), wherein contamination with suchribonucleases may sometimes completely degrade RNA in solution.Accordingly, the natural degradation of mRNA in the cytoplasm of cellsis very finely regulated and RNase contaminations may be generallyremoved by special treatment prior to use of said compositions, inparticular with diethyl pyrocarbonate (DEPC). A number of mechanisms ofnatural degradation are known in this connection in the prior art, whichmay be utilized as well. E.g., the terminal structure is typically ofcritical importance for a mRNA in vivo. As an example, at the 5′ end ofnaturally occurring mRNAs there is usually a so-called “cap structure”(a modified guanosine nucleotide), and at the 3′ end is typically asequence of up to 200 adenosine nucleotides (the so-called poly-A tail).

The at least one RNA of the active (immunostimulatory) composition ofthe present invention, particularly if provided as a mRNA, can thereforebe stabilized against degradation by RNases by the addition of aso-called “5′ cap” structure. Particular preference is given in thisconnection to an m7G(5′)ppp (5′(A,G(5′)ppp(5′)A or G(5′)ppp(5′)G as the5′ cap” structure. However, such a modification is introduced only if amodification, for example a lipid modification, has not already beenintroduced at the 5′ end of the (m)RNA of the inventiveimmunostimulatory composition or if the modification does not interferewith the immunogenic properties of the (unmodified or chemicallymodified) (m)RNA.

According to a further preferred embodiment, the at least one RNA of theactive (immunostimulatory) composition of the present invention maycontain, especially if the RNA is in the form of a mRNA, a poly-A tailon the 3′ terminus of typically about 10 to 200 adenosine nucleotides,preferably about 10 to 100 adenosine nucleotides, more preferably about20 to 100 adenosine nucleotides or even more preferably about 40 to 80adenosine nucleotides.

According to a further preferred embodiment, the at least one RNA of theactive (immunostimulatory) composition of the present invention maycontain, especially if the RNA is in the form of a mRNA, a poly-C tailon the 3′ terminus of typically about 10 to 200 cytosine nucleotides,preferably about 10 to 100 cytosine nucleotides, more preferably about20 to 70 cytosine nucleotides or even more preferably about 20 to 60 oreven 10 to 40 cytosine nucleotides.

According to another embodiment, the at least one RNA of the active(immunostimulatory) composition of the present invention may bemodified, and thus stabilized, especially if the RNA is in the form of amRNA, by modifying the G/C content of the RNA, preferably of the codingregion of the at least one RNA.

In a particularly preferred embodiment of the present invention, the G/Ccontent of the coding region of the at least one (m)RNA of the active(immunostimulatory) composition of the present invention is modified,particularly increased, compared to the G/C content of the coding regionof its particular wild-type (m)RNA, i.e. the unmodified (m)RNA. Theencoded amino acid sequence of the at least one (m)RNA is preferably notmodified compared to the coded amino acid sequence of the particularwild-type (m)RNA.

This modification of the at least one (m)RNA of the active(immunostimulatory) composition of the present invention is based on thefact that the sequence of any (m)RNA region to be translated isimportant for efficient translation of that (m)RNA. Thus, thecomposition and the sequence of various nucleotides is important. Inparticular, sequences having an increased G (guanosine)/C (cytosine)content are more stable than sequences having an increased A(adenosine)/U (uracil) content. According to the invention, the codonsof the (m)RNA are therefore varied compared to its wild-type (m)RNA,while retaining the translated amino acid sequence, such that theyinclude an increased amount of G/C nucleotides. In respect to the factthat several codons code for one and the same amino acid (so-calleddegeneration of the genetic code), the most favorable codons for thestability can be determined (so-called alternative codon usage).

Depending on the amino acid to be encoded by the at least one (m)RNA,there are various possibilities for modification of the at least one(m)RNA sequence, compared to its wild-type sequence. In the case ofamino acids which are encoded by codons which contain exclusively G or Cnucleotides, no modification of the codon is necessary. Thus, the codonsfor Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC orGGG) require no modification, since no A or U is present.

In contrast, codons which contain A and/or U nucleotides can be modifiedby substitution of other codons which code for the same amino acids butcontain no A and/or U. Examples of these are:

the codons for Pro can be modified from CCU or CCA to CCC or CCG;

the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGCor CGG;

the codons for Ala can be modified from GCU or GCA to GCC or GCG;

the codons for Gly can be modified from GGU or GGA to GGC or GGG.

In other cases, although A or U nucleotides cannot be eliminated fromthe codons, it is however possible to decrease the A and U content byusing codons which contain a lower content of A and/or U nucleotides.Examples of these are:

the codons for Phe can be modified from UUU to UUC;

the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC orCUG;

the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG orAGC;

the codon for Tyr can be modified from UAU to UAC;

the codon for Cys can be modified from UGU to UGC;

the codon for His can be modified from CAU to CAC;

the codon for Gln can be modified from CAA to CAG;

the codons for Ile can be modified from AUU or AUA to AUC;

the codons for Thr can be modified from ACU or ACA to ACC or ACG;

the codon for Asn can be modified from AAU to AAC;

the codon for Lys can be modified from AAA to AAG;

the codons for Val can be modified from GUU or GUA to GUC or GUG;

the codon for Asp can be modified from GAU to GAC;

the codon for Glu can be modified from GAA to GAG;

the stop codon UAA can be modified to UAG or UGA.

In the case of the codons for Met (AUG) and Trp (UGG), on the otherhand, there is no possibility of sequence modification.

The substitutions listed above can be used either individually or in allpossible combinations to increase the G/C content of the at least one(m)RNA of the active (immunostimulatory) composition of the presentinvention compared to its particular wild-type (m)RNA (i.e. the originalsequence). Thus, for example, all codons for Thr occurring in thewild-type sequence can be modified to ACC (or ACG). Preferably, however,for example, combinations of the above substitution possibilities areused:

substitution of all codons coding for Thr in the original sequence(wild-type (m)RNA) to ACC (or ACG) and

substitution of all codons originally coding for Ser to UCC (or UCG orAGC);

substitution of all codons coding for Ile in the original sequence toAUC and

substitution of all codons originally coding for Lys to AAG and

substitution of all codons originally coding for Tyr to UAC;

substitution of all codons coding for Val in the original sequence toGUC (or GUG) and

substitution of all codons originally coding for Glu to GAG and

substitution of all codons originally coding for Ala to GCC (or GCG) and

substitution of all codons originally coding for Arg to CGC (or CGG);

substitution of all codons coding for Val in the original sequence toGUC (or GUG) and

substitution of all codons originally coding for Glu to GAG and

substitution of all codons originally coding for Ala to GCC (or GCG) and

substitution of all codons originally coding for Gly to GGC (or GGG) and

substitution of all codons originally coding for Asn to AAC;

substitution of all codons coding for Val in the original sequence toGUC (or GUG) and

substitution of all codons originally coding for Phe to UUC and

substitution of all codons originally coding for Cys to UGC and

substitution of all codons originally coding for Leu to CUG (or CUC) and

substitution of all codons originally coding for Gln to CAG and

substitution of all codons originally coding for Pro to CCC (or CCG);etc.

Preferably, the G/C content of the coding region of the at least one(m)RNA of the active (immunostimulatory) composition of the presentinvention is increased by at least 7%, more preferably by at least 15%,particularly preferably by at least 20%, compared to the G/C content ofthe coded region of the wild-type (m)RNA which codes for an antigen,antigenic protein or antigenic peptide as determined herein or itsfragment or variant thereof. According to a specific embodiment at least5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, evenmore preferably at least 80% and most preferably at least 90%, 95% oreven 100% of the substitutable codons in the region coding for anantigen, antigenic protein or antigenic peptide as deinined herein orits fragment or variant thereof or the whole sequence of the wild type(m)RNA sequence are substituted, thereby increasing the GC/content ofsaid sequence.

In this context, it is particularly preferable to increase the G/Ccontent of the at least one (m)RNA of the active (immunostimulatory)composition of the present invention to the maximum (i.e. 100% of thesubstitutable codons), in particular in the region coding for a protein,compared to the wild-type sequence.

According to the invention, a further preferred modification of the atleast one (m)RNA of the active (immunostimulatory) composition of thepresent invention is based on the finding that the translationefficiency is also determined by a different frequency in the occurrenceof tRNAs in cells. Thus, if so-called “rare codons” are present in theat least one (m)RNA of the active (immunostimulatory) composition of thepresent invention to an increased extent, the corresponding modified atleast one (m)RNA sequence is translated to a significantly poorer degreethan in the case where codons coding for relatively “frequent” tRNAs arepresent.

According to the invention, in the modified at least one (m)RNA of theactive (immunostimulatory) composition of the present invention, theregion which codes for the adjuvant protein is modified compared to thecorresponding region of the wild-type (m)RNA such that at least onecodon of the wild-type sequence which codes for a tRNA which isrelatively rare in the cell is exchanged for a codon which codes for atRNA which is relatively frequent in the cell and carries the same aminoacid as the relatively rare tRNA. By this modification, the sequences ofthe at least one (m)RNA of the active (immunostimulatory) composition ofthe present invention is modified such that codons for which frequentlyoccurring tRNAs are available are inserted. In other words, according tothe invention, by this modification all codons of the wild-type sequencewhich code for a tRNA which is relatively rare in the cell can in eachcase be exchanged for a codon which codes for a tRNA which is relativelyfrequent in the cell and which, in each case, carries the same aminoacid as the relatively rare tRNA.

Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly codon, which uses the tRNA which occursthe most frequently in the (human) cell, are particularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe modified at least one (m)RNA of the active (immunostimulatory)composition of the present invention, with the “frequent” codons withoutmodifying the amino acid sequence of the protein encoded by the codingregion of the (m)RNA. This preferred embodiment allows provision of aparticularly efficiently translated and stabilized (modified) at leastone (m)RNA of the active (immunostimulatory) composition of the presentinvention.

The determination of a modified at least one (m)RNA of the active(immunostimulatory) composition of the present invention as describedabove (increased G/C content; exchange of tRNAs) can be carried outusing the computer program explained in WO 02/098443—the disclosurecontent of which is included in its full scope in the present invention.Using this computer program, the nucleotide sequence of any desired(m)RNA can be modified with the aid of the genetic code or thedegenerative nature thereof such that a maximum G/C content results, incombination with the use of codons which code for tRNAs occurring asfrequently as possible in the cell, the amino acid sequence coded by themodified at least one (m)RNA preferably not being modified compared tothe non-modified sequence. Alternatively, it is also possible to modifyonly the G/C content or only the codon usage compared to the originalsequence. The source code in Visual Basic 6.0 (development environmentused: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is alsodescribed in WO 02/098443.

In a further preferred embodiment of the present invention, the A/Ucontent in the environment of the ribosome binding site of the at leastone (m)RNA of the active (immunostimulatory) composition of the presentinvention is increased compared to the A/U content in the environment ofthe ribosome binding site of its particular wild-type (m)RNA. Thismodification (an increased A/U content around the ribosome binding site)increases the efficiency of ribosome binding to the at least one (m)RNA.An effective binding of the ribosomes to the ribosome binding site(Kozak sequence: GCCGCCACCAUGG (SEQ ID NO: 27), the AUG forms the startcodon) in turn has the effect of an efficient translation of the atleast one (m)RNA.

According to a further embodiment of the present invention the at leastone (m)RNA of the active (immunostimulatory) composition of the presentinvention may be modified with respect to potentially destabilizingsequence elements. Particularly, the coding region and/or the 5′ and/or3′ untranslated region of this at least one (m)RNA may be modifiedcompared to the particular wild-type (m)RNA such that is contains nodestabilizing sequence elements, the coded amino acid sequence of themodified at least one (m)RNA preferably not being modified compared toits particular wild-type (m)RNA. It is known that, for example, insequences of eukaryotic RNAs destabilizing sequence elements (DSE)occur, to which signal proteins bind and regulate enzymatic degradationof RNA in vivo. For further stabilization of the modified at least one(m)RNA, optionally in the region which encodes for an antigen, antigenicprotein or antigenic peptide as defined herein, one or more suchmodifications compared to the corresponding region of the wild-type(m)RNA can therefore be carried out, so that no or substantially nodestabilizing sequence elements are contained there. According to theinvention, DSE present in the untranslated regions (3′- and/or 5′-UTR)can also be eliminated from the at least one (m)RNA of the active(immunostimulatory) composition of the present invention by suchmodifications.

Such destabilizing sequences are e.g. AU-rich sequences (AURES), whichoccur in 3′-UTR sections of numerous unstable RNAs (Caput et at, Proc.Natl. Acad. Sci. USA 1986, 83: 1670 to 1674). The at least one (m)RNA ofthe active (immunostimulatory) composition of the present invention istherefore preferably modified compared to the wild-type (m)RNA such thatthe at least one (m)RNA contains no such destabilizing sequences. Thisalso applies to those sequence motifs which are recognized by possibleendonucleases, e.g. the sequence GAACAAG, which is contained in the3′-UTR segment of the gene which codes for the transferrin receptor(Binder et at, EMBO J. 1994, 13: 1969 to 1980). These sequence motifsare also preferably removed in the at least one (m)RNA of the active(immunostimulatory) composition of the present invention.

Also preferably according to the invention, the at least one (m)RNA ofthe active (immunostimulatory) composition of the present invention has,in a modified form, at least one IRES as defined above and/or at leastone 5′ and/or 3′ stabilizing sequence, in a modified form, e.g. toenhance ribosome binding or to allow expression of different encodedantigens located on an at least one (bi- or even multicistronic) RNA ofthe active (immunostimulatory) composition of the present invention.

According to the invention, the at least one (m)RNA of the active(immunostimulatory) composition of the present invention furthermorepreferably has at least one 5′ and/or 3′ stabilizing sequence. Thesestabilizing sequences in the 5′ and/or 3′ untranslated regions have theeffect of increasing the half-life of the at least one (m)RNA in thecytosol. These stabilizing sequences can have 100% sequence homology tonaturally occurring sequences which occur in viruses, bacteria andeukaryotes, but can also be partly or completely synthetic. Theuntranslated sequences (UTR) of the globin gene, e.g. from Homo sapiensor Xenopus laevis may be mentioned as an example of stabilizingsequences which can be used in the present invention for a stabilizedRNA. Another example of a stabilizing sequence has the general formula(C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO: 28), which is containedin the 3′UTR of the very stable RNA which codes for globin,(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf. Holcik etal., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Suchstabilizing sequences can of course be used individually or incombination with one another and also in combination with otherstabilizing sequences known to a person skilled in the art. The at leastone (m)RNA of the active (immunostimulatory) composition of the presentinvention is therefore preferably present as globin UTR (untranslatedregions)-stabilized RNA, in particular as globin UTR-stabilized RNA.

Nevertheless, substitutions, additions or eliminations of bases arepreferably carried out with the at least one RNA of the active(immunostimulatory) composition of the present invention, using a DNAmatrix for preparation of the at least one RNA of the active(immunostimulatory) composition of the present invention by techniquesof the well known site directed mutagenesis or with an oligonucleotideligation strategy (see e.g. Maniatis et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., ColdSpring Harbor, NY, 2001). In such a process, for preparation of the atleast one (m)RNA, a corresponding DNA molecule may be transcribed invitro. This DNA matrix preferably comprises a suitable promoter, e.g. aT7 or SP6 promoter, for in vitro transcription, which is followed by thedesired nucleotide sequence for the at least one RNA to be prepared anda termination signal for in vitro transcription. The DNA molecule, whichforms the matrix of an at least one RNA of interest, may be prepared byfermentative proliferation and subsequent isolation as part of a plasmidwhich can be replicated in bacteria. Plasmids which may be mentioned assuitable for the present invention are e.g. the plasmids pT7Ts (GenBankaccession number U26404; Lai et al, Development 1995, 121: 2349 to2360), pGEM® series, e.g. pGEM®-1 (GenBank accession number X65300; fromPromega) and pSP64 (GenBank accession number X65327); cf. also Mezei andStorts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCRTechnology: Current Innovation, CRC Press, Boca Raton, Fla., 2001.

The stabilization of the at least one RNA of the active(immunostimulatory) composition of the present invention can likewise bycarried out by associating or complexing the at least one RNA with, orbinding it to, a cationic compound, in particular a polycationiccompound, for example a (poly)cationic peptide or protein. In particularthe use of protamine, nucleoline, spermin or spermidine as thepolycationic, nucleic-acid-binding protein to the RNA is particularlyeffective. Furthermore, the use of other cationic peptides or proteins,such as poly-L-lysine or histones, is likewise possible. This procedurefor stabilizing RNA is described in EP-A-1083232, the disclosure ofwhich is incorporated by reference into the present invention in itsentirety. Further preferred cationic substances which can be used forstabilizing the RNA of the active (immunostimulatory) composition of thepresent invention include cationic polysaccharides, for examplechitosan, polybrene, polyethyleneimine (PEI) or poly-L-lysine (PLL),etc. Association or complexing of the at least one RNA of the inventiveactive (immunostimulatory) composition with cationic compounds, e.g.cationic proteins or cationic lipids, e.g. oligofectamine as a lipidbased complexation reagent) preferably increases the transfer of the atleast one RNA present as a pharmaceutically active component into thecells to be treated or into the organism to be treated. It is alsoreferred to the disclosure herein with regard to the stabilizing effectfor the at least one RNA of the active (immunostimulatory) compositionof the present invention by complexation, which holds for thestabilization of RNA as well.

According to another particularly preferred embodiment, the at least RNAof the active (immunostimulatory) composition may additionally oralternatively encode a secretory signal peptide. Such signal peptidesare sequences, which typically exhibit a length of about 15 to 30 aminoacids and are preferably located at the N-terminus of the encodedpeptide, without being limited thereto. Signal peptides as definedherein preferably allow the transport of the antigen, antigenic proteinor antigenic peptide as encoded by the at least one RNA of the active(immunostimulatory) composition into a defined cellular compartment,preferably the cell surface, the endoplasmic reticulum (ER) or theendosomal-lysosomal compartment. Examples of secretory signal peptidesequences as defined herein include, without being limited thereto,signal sequences of classical or non-classical MHC-molecules (e.g.signal sequences of MHC I and II molecules, e.g. of the MHC class Imolecule HLA-A*0201), signal sequences of cytokines or immunoglobulinesas defined herein, signal sequences of the invariant chain ofimmunoglobulines or antibodies as defined herein, signal sequences ofLampl, Tapasin, Erp57, Calretikulin, Calnexin, and further membraneassociated proteins or of proteins associated with the endoplasmicreticulum (ER) or the endosomal-lysosomal compartment. Particularlypreferably, signal sequences of MHC class I molecule HLA-A*0201 may beused according to the present invention.

Any of the above modifications may be applied to the at least one RNA ofthe active (immunostimulatory) composition of the present invention, andfurther to any (m)RNA as used in the context of the present inventionand may be, if suitable or necessary, be combined with each other in anycombination, provided, these combinations of modifications do notinterfere with each other in the respective at least one RNA. A personskilled in the art will be able to take his choice accordingly.

According to another embodiment, the active (immunostimulatory)composition according to the invention may comprise an adjuvant. In thiscontext, an adjuvant may be understood as any compound, which issuitable to support administration and delivery of the active(immunostimulatory) composition according to the invention. Furthermore,such an adjuvant may, without being bound thereto, initiate or increasean immune response of the innate immune system, i.e. a non-specificimmune response. With other words, when administered, the active(immunostimulatory) composition according to the invention typicallyinitiates an adaptive immune response due to the at least two antigensencoded by the at least one RNA contained in the inventive active(immunostimulatory) composition. Additionally, the active(immunostimulatory) composition according to the invention may generatean (supportive) innate immune response due to addition of an adjuvant asdefined herein to the active (immunostimulatory) composition accordingto the invention. Such an adjuvant may be selected from any adjuvantknown to a skilled person and suitable for the present case, i.e.supporting the induction of an immune response in a mammal. Preferably,the adjuvant may be selected from the group consisting of, without beinglimited thereto, TDM, MDP, muramyl dipeptide, pluronics, alum solution,aluminium hydroxide, ADJUMER™ (polyphosphazene); aluminium phosphategel; glucans from algae; algammulin; aluminium hydroxide gel (alum);highly protein-adsorbing aluminium hydroxide gel; low viscosityaluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80(0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4);AVRIDINE™ (propanediamine); BAY R1005™((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAPTM (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP),ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline saltcomplex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine);ImmTher™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferon-gamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3.™;liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles ofany composition; MF59™; (squalene-water emulsion); MONTANIDE ISA 51™(purified incomplete Freund's adjuvant); MONTANIDE ISA 720™(metabolizable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH₃); MURAPALMITINE™and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles ofany composition; NISVs (non-ionic surfactant vesicles); PLEURAN™(β-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid andglycolic acid; microspheres/nanospheres); PLURONIC L121™; PMMA(polymethyl methacrylate); PODDS™ (proteinoid microspheres);polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylicacid-polyuridylic acid complex); polysorbate 80 (Tween 80); proteincochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™(QS-21); Quil-A (Quil-A saponin); S-28463(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol);SAF-1™ (“Syntex adjuvant formulation”); Sendai proteoliposomes andSendai-containing lipid matrices; Span-85 (sorbitan trioleate); Specol(emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane®(2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);stearyltyrosine(octadecyltyrosine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIORAL; plant derived adjuvants, including QS21, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin,; microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR 1-10, ligands of murineTLR 1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine,IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPIanchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusionprotein, cdiGMP; and adjuvants suitable as antagonists including CGRPneuropeptide.

Suitable adjuvants may also be selected from cationic or polycationiccompounds wherein the adjuvant is preferably prepared upon complexingthe at least one RNA of the inventive active (immmunostimulatorycomposition) with the cationic or polycationic compound. Association orcomplexing the RNA of the active (immunostimulatory) composition withcationic or polycationic compounds as defined herein preferably providesadjuvant properties and confers a stabilizing effect to the at least oneRNA of the active (immunostimulatory) composition. Particularly suchpreferred, such cationic or polycationic compounds are selected fromcationic or polycationic peptides or proteins, including protamine,nucleoline, spermin or spermidine, or other cationic peptides orproteins, such as poly-L-lysine (PLL), poly-arginine, basicpolypeptides, cell penetrating peptides (CPPs), including HIV-bindingpeptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA orprotein transduction domains (PTDs, PpT620, prolin-rich peptides,arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1,L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, plsl, FGF,Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC,hCT-derived peptides, SAP, protamine, spermine, spermidine, or histones.Further preferred cationic or polycationic compounds may includecationic polysaccharides, for example chitosan, polybrene, cationicpolymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, Chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,Blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected of a cationic polymer as mentioned above) and of one ormore hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole); etc.

Additionally, preferred cationic or polycationic proteins or peptides,which can be used as an adjuvant by complexing the at least one RNA ofthe active (immunostimulatory) composition, may be selected fromfollowing proteins or peptides having the following total formula (I):(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), whereinl+m+n+o+x=8-15, and l, m, n, or o independently of each other may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred oligoarginines in this context are e.g. Arg₇,Arg₈, Arg₉, Arg₇, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc.

Suitable adjuvants may furthermore be selected from nucleic acids havingthe formula (II): G_(l)X_(m)G_(n), wherein: G is guanosine, uracil or ananalogue of guanosine or uracil; X is guanosine, uracil, adenosine,thymidine, cytosine or an analogue of the above-mentioned nucleotides; lis an integer from 1 to 40, wherein when l=1 G is guanosine or ananalogue thereof, when l>1 at least 50% of the nucleotides are guanosineor an analogue thereof; m is an integer and is at least 3; wherein whenm=3 X is uracil or an analogue thereof, when m>3 at least 3 successiveuracils or analogues of uracil occur; n is an integer from 1 to 40,wherein when n=1 G is guanosine or an analogue thereof, when n>1 atleast 50% of the nucleotides are guanosine or an analogue thereof.

Other suitable adjuvants may furthermore be selected from nucleic acidshaving the formula (III): C_(l)X_(m)C_(n), wherein: C is cytosine,uracil or an analogue of cytosine or uracil; X is guanosine, uracil,adenosine, thymidine, cytosine or an analogue of the above-mentionednucleotides; l is an integer from 1 to 40, wherein when l=1 C iscytosine or an analogue thereof, when l>1 at least 50% of thenucleotides are cytosine or an analogue thereof; m is an integer and isat least 3; wherein when m=3 X is uracil or an analogue thereof, whenm>3 at least 3 successive uracils or analogues of uracil occur; n is aninteger from 1 to 40, wherein when n=1 C is cytosine or an analoguethereof, when n>1 at least 50% of the nucleotides are cytosine or ananalogue thereof.

According to one preferred embodiment, the present invention mayfurthermore provide a vaccine containing the active (immunostimulatory)composition according to the invention. The inventive vaccine mayadditionally contain a pharmaceutically acceptable carrier and/orfurther auxiliary substances and additives and/or adjuvants. Accordingto a particularly preferred embodiment, the antigens encoded by the atleast one RNA of the active (immunostimulatory) composition, containedin the inventive vaccine, are selected from the above mentioned groupsor subgroups. According to an even more preferred embodiment, theprotein antigens are selected from any of the antigens of the followingsubgroup comprising NY-ESO1[accession number NM_(—)001327], hTERT[accession number NM_(—)198253], survivin [accession number AF077350],5T4 [accession number NM_(—)006670] and WT1 [accession numberNM_(—)000378], and/or from any of the antigens of the following subgroupcomprising MAGE-C1 and MAGE-C2, as defined herein, and/or from any ofthe antigens of the following subgroup comprising MAGE-A2 and MAGE-A3,as defined herein.

The inventive vaccine typically comprises a safe and effective amount ofthe at least one RNA of the active (immunostimulatory) composition asdefined above encoding at least two antigens as defined above, morepreferably encoding at least two antigens selected from any of the abovegroups or subgroups, most preferably in any of the indicatedcombinations. As used herein, “safe and effective amount” means anamount of the at least one RNA of the active (immunostimulatory)composition in the vaccine as defined above, that is sufficient tosignificantly induce a positive modification of lung cancer, preferablyof a non-small-cell lung cancer (NSCLC) related condition to be treated,more preferably of conditions related to the three main sub-types ofnon-small-cell lung cancer (NSCLC) including, without being restrictedthereto, squamous cell lung carcinoma, adenocarcinoma and large celllung carcinoma. At the same time, however, a “safe and effective amount”is small enough to avoid serious side-effects, that is to say to permita sensible relationship between advantage and risk. The determination ofthese limits typically lies within the scope of sensible medicaljudgment. In relation to the inventive vaccine, the expression “safe andeffective amount” preferably means an amount of the RNA (and thus of theencoded at least two antigens) that is suitable for stimulating theadaptive immune system in such a manner that no excessive or damagingimmune reactions are achieved but, preferably, also no such immunereactions below a measurable level. Such a “safe and effective amount”of the at least one RNA of the active (immunostimulatory) composition inthe vaccine as defined above may furthermore be selected in dependenceof the type of RNA, e.g. monocistronic, bi- or even multicistronic RNA,since a bi- or even multicistronic RNA may lead to a significantlyhigher expression of the encoded antigen(s) than use of an equal amountof a monocistronic RNA. A “safe and effective amount” of the at leastone RNA of the active (immunostimulatory) composition as defined above,which is contained in the inventive vaccine, will furthermore vary inconnection with the particular condition to be treated and also with theage and physical condition of the patient to be treated, the severity ofthe condition, the duration of the treatment, the nature of theaccompanying therapy, of the particular pharmaceutically acceptablecarrier used, and similar factors, within the knowledge and experienceof the accompanying doctor. The vaccine according to the invention canbe used according to the invention for human and also for veterinarymedical purposes, as a pharmaceutical composition or as a vaccine.

The vaccine according to the invention typically contains apharmaceutically acceptable carrier. The expression “pharmaceuticallyacceptable carrier” as used herein preferably includes the liquid ornon-liquid basis of the inventive vaccine. If the inventive vaccine isprovided in liquid form, the carrier will typically be pyrogen-freewater; isotonic saline or buffered (aqueous) solutions, e.g. phosphate-,citrate-buffered solutions, etc. Particularly for injection of theinventive vaccine, water or preferably a buffer, more preferably anaqueous buffer, may be used, containing a sodium salt, preferably atleast 50 mM of a sodium salt, a calcium salt, preferably at least 0.01mM of a calcium salt, and optionally a potassium salt, preferably atleast 3 mM of a potassium salt. According to a preferred embodiment, thesodium, calcium and, optionally, potassium salts may occur in the formof their halogenides, e.g. chlorides, iodides, or bromides, in the formof their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.Without being limited thereto, examples of sodium salts include e.g.NaCl, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄, examples of the optionalpotassium salts include e.g. KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, andexamples of calcium salts include e.g. CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄,Ca(OH)₂. Furthermore, organic anions of the aforementioned cations maybe contained in the buffer. According to a more preferred embodiment,the buffer suitable for injection purposes as defined above, may containsalts selected from sodium chloride (NaCl), calcium chloride (CaCl₂) andoptionally potassium chloride (KCl), wherein further anions may bepresent additional to the chlorides. CaCl₂ can also be replaced byanother salt like KCl. Typically, the salts in the injection buffer arepresent in a concentration of at least 50 mM sodium chloride (NaCl), atleast 3 mM potassium chloride (KCl) and at least 0.01 mM calciumchloride (CaCl₂). The injection buffer may be hypertonic, isotonic orhypotonic with reference to the specific reference medium, i.e. thebuffer may have a higher, identical or lower salt content with referenceto the specific reference medium, wherein preferably such concentrationsof the afore mentioned salts may be used, which do not lead to damage ofcells due to osmosis or other concentration effects. Reference media aree.g. in “in vivo” methods occurring liquids such as blood, lymph,cytosolic liquids, or other body liquids, or e.g. liquids, which may beused as reference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well, which are suitable foradministration to a person. The term “compatible” as used herein meansthat the constituents of the inventive vaccine are capable of beingmixed with the at least one RNA of the active (immunostimulatory)composition, encoding at least two antigens as defined above, in such amanner that no interaction occurs which would substantially reduce thepharmaceutical effectiveness of the inventive vaccine under typical useconditions. Pharmaceutically acceptable carriers, fillers and diluentsmust, of course, have sufficiently high purity and sufficiently lowtoxicity to make them suitable for administration to a person to betreated. Some examples of compounds which can be used aspharmaceutically acceptable carriers, fillers or constituents thereofare sugars, such as, for example, lactose, glucose and sucrose;starches, such as, for example, corn starch or potato starch; celluloseand its derivatives, such as, for example, sodiumcarboxymethylcellulose, ethylcellulose, cellulose acetate; powderedtragacanth; malt; gelatin; tallow; solid glidants, such as, for example,stearic acid, magnesium stearate; calcium sulfate; vegetable oils, suchas, for example, groundnut oil, cottonseed oil, sesame oil, olive oil,corn oil and oil from theobroma; polyols, such as, for example,polypropylene glycol, glycerol, sorbitol, mannitol and polyethyleneglycol; alginic acid.

The choice of a pharmaceutically acceptable carrier is determined inprinciple by the manner in which the inventive vaccine is administered.The inventive vaccine can be administered, for example, systemically orlocally. Routes for systemic administration in general include, forexample, transdermal, oral, parenteral routes, including subcutaneous,intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,vaccines may be administered by an intradermal, subcutaneous, orintramuscular route. Compositions/vaccines are therefore preferablyformulated in liquid or solid form. The suitable amount of the inventivevaccine to be administered can be determined by routine experiments withanimal models. Such models include, without implying any limitation,rabbit, sheep, mouse, rat, dog and non-human primate models. Preferredunit dose forms for injection include sterile solutions of water,physiological saline or mixtures thereof. The pH of such solutionsshould be adjusted to about 7.4. Suitable carriers for injection includehydrogels, devices for controlled or delayed release, polylactic acidand collagen matrices. Suitable pharmaceutically acceptable carriers fortopical application include those which are suitable for use in lotions,creams, gels and the like. If the inventive vaccine is to beadministered perorally, tablets, capsules and the like are the preferredunit dose form. The pharmaceutically acceptable carriers for thepreparation of unit dose forms which can be used for oral administrationare well known in the prior art. The choice thereof will depend onsecondary considerations such as taste, costs and storability, which arenot critical for the purposes of the present invention, and can be madewithout difficulty by a person skilled in the art.

The inventive vaccine can additionally contain one or more auxiliarysubstances in order to further increase the immunogenicity. Asynergistic action of the at least one RNA of the active(immunostimulatory) composition as defined above and of an auxiliarysubstance, which may be optionally also contained in the inventivevaccine as described above, is preferably achieved thereby. Depending onthe various types of auxiliary substances, various mechanisms can comeinto consideration in this respect. For example, compounds that permitthe maturation of dendritic cells (DCs), for examplelipopolysaccharides, TNF-alpha or CD40 ligand, form a first class ofsuitable auxiliary substances. In general, it is possible to use asauxiliary substance any agent that influences the immune system in themanner of a “danger signal” (LPS, GP96, etc.) or cytokines, such asGM-CFS, which allow an immune response produced by theimmune-stimulating adjuvant according to the invention to be enhancedand/or influenced in a targeted manner. Particularly preferred auxiliarysubstances are cytokines, such as monokines, lymphokines, interleukinsor chemokines, that—additional to induction of the adaptive immuneresponse by the encoded at least two antigens—promote the innate immuneresponse, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30,IL-31, IL-32, IL-33, INF-alpha, IFN-beta, INF-gamma, GM-CSF,G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.

Further additives which may be included in the inventive vaccine areemulsifiers, such as, for example, Tween®; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

The inventive vaccine can also additionally contain any furthercompound, which is known to be immune-stimulating due to its bindingaffinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which may be added to an inventive vaccinein this context, may be CpG nucleic acids, in particular CpG-RNA orCpG-DNA. A CpG-RNA or CpG-DNA can be a single-stranded CpG-DNA (ssCpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA(ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleicacid is preferably in the form of CpG-RNA, more preferably in the formof single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid preferablycontains at least one or more (mitogenic) cytosine/guanine dinucleotidesequence(s) (CpG motif(s)). According to a first preferred alternative,at least one CpG motif contained in these sequences, that is to say theC (cytosine) and the G (guanine) of the CpG motif, is unmethylated. Allfurther cytosines or guanines optionally contained in these sequencescan be either methylated or unmethylated. According to a furtherpreferred alternative, however, the C (cytosine) and the G (guanine) ofthe CpG motif can also be present in methylated form.

According to a further preferred object of the present invention, theinventive active (immunostimulatory) composition or the at least one RNAencoding at least two (preferably) different antigens as defined herein,may be used (for the preparation of a vaccine according to the presentinvention) for the treatment of lung cancer, preferably of anon-small-cell lung cancer (NSCLC) related condition, more preferably ofconditions related to the three main sub-types of non-small-cell lungcancer (NSCLC) including, without being restricted thereto, squamouscell lung carcinoma, adenocarcinoma and large cell lung carcinoma.

According to a further preferred object of the present invention, theinventive vaccine or the at least one RNA encoding at least two(preferably) different antigens as defined herein may be used for thetreatment of lung cancer, preferably of a non-small-cell lung cancer(NSCLC) related condition, more preferably of conditions related to thethree main sub-types of non-small-cell lung cancer (NSCLC) including,without being restricted thereto, squamous cell lung carcinoma,adenocarcinoma and large cell lung carcinoma.

In this context also included in the present invention are methods oftreating lung cancer, preferably of a non-small-cell lung cancer (NSCLC)related condition, more preferably of conditions related to the threemain sub-types of non-small-cell lung cancer (NSCLC) including, withoutbeing restricted thereto, squamous cell lung carcinoma, adenocarcinomaand large cell lung carcinoma, by administering to a patient in needthereof a pharmaceutically effective amount of an inventive vaccine, ora pharmaceutically effective amount of an inventive active(immunostimulatory) composition. Such a method typically comprises anoptional first step of preparing the inventive active(immunostimulatory) composition, or the inventive vaccine, and a secondstep, comprising administering (a pharmaceutically effective amount of)said inventive active (immunostimulatory) composition or said inventivevaccine to a patient in need thereof. A patient in need thereof will betypically selected from any mammal. In the context of the presentinvention, a mammal is preferably selected from the group comprising,without being limited thereto, e.g. goat, cattle, swine, dog, cat,donkey, monkey, ape, a rodent such as a mouse, hamster, rabbit and,particularly, human, wherein the mammal typically suffers from lungcancer, preferably of a non-small-cell lung cancer (NSCLC) relatedcondition, more preferably of conditions related to the three mainsub-types of non-small-cell lung cancer (NSCLC) including, without beingrestricted thereto, squamous cell lung carcinoma, adenocarcinoma andlarge cell lung carcinoma or a condition related thereto.

The invention relates also to the use of the inventive active(immunostimulatory) composition or the at least one RNA encoding atleast two (preferably) different antigens as defined herein (for thepreparation of an inventive vaccine), preferably for eliciting an immuneresponse in a mammal, preferably for the treatment of lung cancer, morepreferably for the treatment of a non-small-cell lung cancer (NSCLC)related condition as defined herein.

Similarly, the invention also relates also to the use of the inventivevaccine per se or the at least one RNA encoding at least two(preferably) different antigens as defined herein for eliciting anadaptive immune response in a mammal, preferably for the treatment oflung cancer, more preferably of a non-small-cell lung cancer (NSCLC)related condition as defined herein.

Prevention or treatment of lung cancer in a patient in need thereof,preferably of a non-small-cell lung cancer (NSCLC) related condition asdefined herein, may be carried out by administering the inventive active(immunostimulatory) composition and/or the inventive vaccine at once orin a time staggered manner, e.g. as a kit of parts, each part containingat least one preferably different antigen. For administration,preferably any of the administration routes may be used as definedabove. E.g., one may treat lung cancer, preferably a non-small-cell lungcancer (NSCLC) related condition as defined herein, by inducing orenhancing an adaptive immune response on the basis of at least two(specifically selected) antigens encoded by the at least one RNA of theinventive active (immunostimulatory) composition. Administering of theinventive active (immunostimulatory) composition and/or the inventivevaccine may then occur prior, concurrent and/or subsequent toadministering another inventive inventive active (immunostimulatory)composition and/or inventive vaccine as defined herein which may containanother combination of RNAs encoding different antigens, wherein eachantigen encoded by the at least one RNA of the inventive active(immunostimulatory) composition may preferably be suitable for thetherapy of lung cancer, more preferably for the treatment of anon-small-cell lung cancer (NSCLC) related condition as defined herein.In this context, a therapy as defined herein may also comprise themodulation of a disease associated to lung cancer, preferably a diseaseassociated to non-small-cell lung cancer (NSCLC) as defined herein.

According to one further embodiment, the present invention furthermorecomprises the use of the active (immunostimulatory) composition (for thepreparation of an (inventive) vaccine) for modulating, preferably toinduce or enhance, an immune response in a mammal as defined above, morepreferably to support the treatment of lung cancer, especially NSCLC asdefined herein. In this context, support of the treatment of lungcancer, especially NSCLC as defined herein, may be any combination of aconventional cancer therapy for lung cancer, especially for NSCLC asdefined herein, such as radiation therapy, chemotherapy, proton therapy,hormonal therapy, antibody therapy, adjuvant therapies, therapiesincluding other vaccines than an inventive vaccine, therapies includingkinase inhibitors or small nucleotides, etc., or some combination ofthese, and a therapy using the inventive active (immunostimulatory)composition or the inventive vaccine as defined herein. Support of thetreatment of lung cancer, especially NSCLC as defined herein, may bealso envisaged in any of the other embodiments defined herein.

Administration of the inventive active (immunostimulatory) compositionor the at least one RNA encoding at least two (preferably) differentantigens as defined herein or the inventive vaccine may be carried outin a time staggered treatment. A time staggered treatment may be e.g.administration of the inventive active (immunostimulatory) compositionor the at least one RNA encoding at least two (preferably) differentantigens as defined herein or the inventive vaccine prior, concurrentand/or subsequent to a therapy of lung cancer, especially NSCLC, e.g. byadministration of the inventive active (immunostimulatory) compositionor vaccine prior, concurrent and/or subsequent to a therapy or anadministration of a therapeutic suitable for the treatment of lungcancer, especially of NSCLC as defined herein. Such time staggeredtreatment may be carried out using e.g. a kit, preferably a kit of partsas defined below.

Time staggered treatment may additionally or alternatively also comprisean administration of the inventive active (immunostimulatory)composition or vaccine, preferably of the at least one RNA encoding atleast two (preferably different) antigens as defined above, in a form,wherein the at least one RNA encoding at least two (preferablydifferent) antigens as defined above, preferably forming part of theinventive active (immunostimulatory) composition or vaccine, isadministered parallel, prior or subsequent to another at least one RNAencoding at least two (preferably different) antigens as defined above,preferably forming part of the same inventive active (immunostimulatory)composition or vaccine. Preferably, the administration (of all at leastone RNAs) occurs within an hour, more preferably within 30 minutes, evenmore preferably within 15, 10, 5, 4, 3, or 2 minutes or even within 1minute. Such time staggered treatment may be carried out using e.g. akit, preferably a kit of parts as defined below.

According to a final embodiment, the present invention also provideskits, particularly kits of parts, comprising the active inventive(immunostimulatory) composition, and/or the inventive vaccine, andoptionally technical instructions with information on the administrationand dosage of the inventive active (immunostimulatory) compositionand/or the inventive vaccine. The technical instructions may containinformation about administration and dosage of the inventive active(immunostimulatory) composition, and/or the inventive vaccine. Suchkits, preferably kits of parts, may applied e.g. for any of the abovementioned applications or uses, preferably for the use of at least oneinventive active (immunostimulatory) composition (for the preparation ofan inventive vaccine) for the treatment of lung cancer, especially ofNSCLC as defined herein. The kits may also be applied for the use of atleast one inventive active (immunostimulatory) composition (for thepreparation of an inventive vaccine) for the treatment of lung cancer,preferably NSCLC as defined herein, wherein the inventive active(immunostimulatory) composition) and/or the vaccine due to the encodedat least two antigens may be capable to induce or enhance an immuneresponse in a mammal as defined above. Such kits may further be appliedfor the use of at least one inventive active (immunostimulatory)composition, (for the preparation of an inventive vaccine) formodulating, preferably for eliciting, e.g. to induce or enhance, animmune response in a mammal as defined above, and preferably to supporttreatment of lung cancer, especially of NSCLC. Kits of parts, as aspecial form of kits, may contain one or more identical or differentactive inventive (immunostimulatory) compositions and/or one or moreidentical or different inventive vaccines in different parts of the kit.Kits of parts may also contain an (e.g. one) active inventive(immunostimulatory) composition, an (e.g. one) inventive vaccine and/orthe at least one RNA encoding at least one antigen as defined above indifferent parts of the kit, e.g. each part of the kit containing atleast one RNA encoding a preferably different antigen. Additionally, acombination of both types of kits of parts is possible. Kits of partsmay be used, e.g. when a time staggered treatment is envisaged, e.g.when using different formulations and/or increasing concentrations ofthe active inventive (immunostimulatory) composition, the inventivevaccine and/or the at least one RNA encoding at least one antigens asdefined above during the same treatment in vivo. Kits of parts may alsobe used when a separated formulation or administration of the differentantigens of the inventive active (immunostimulatory) composition (i.e.in parts) is envisaged or necessary (e.g. for technical reasons), bute.g. a combined presence of the different antigens in vivo is still tobe achieved. Particularly kits of parts as a special form of kits areenvisaged, wherein each part of the kit contains at least one preferablydifferent antigen as defined above, all parts of the kit of partspreferably forming the active inventive (immunostimulatory) compositionor the inventive vaccine as defined herein. Such specific kits of partsmay particularly be suitable, e.g. if different antigens are formulatedseparately as different parts of the kits, but are then administered atonce together or in a time staggered manner to the mammal in needthereof. In the latter case administration of all of the different partsof such a kit typically occurs within a short time limit, such that allantigens are present in the mammal at about the same time subsequent toadministration of the last part of the kit. Any of the above kits may beused in a treatment as defined above.

Advantages of the Present Invention

The present invention provides an active (immunostimulatory) compositionfor the treatment of lung cancer, particularly of non-small lung cancer(NSCLC), wherein the composition comprises at least one RNA, preferablya mRNA, encoding at least two (preferably different) antigens capable ofeliciting an (adaptive) immune response in a mammal wherein the antigensare selected from the group consisting of hTERT, WT1, MAGE-A2, 5T4,MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1, or MAGE-C2.Such an active (immunostimulatory) composition allows efficienttreatment of lung cancer, particularly of non-small lung cancer (NSCLC),or supplementary treatment when using conventional therapies. Itfurthermore avoids the problem of uncontrolled propagation of theintroduced DNA sequences by the use of RNA as an approach for curativemethods. RNA as used in the inventive active (immunostimulatory)composition has additional considerable advantages over DNA expressionsystems e.g. in immune response, immunization or vaccination. Theseadvantages include, inter alia, that RNA introduced into a cell is notintegrated into the genome. This avoids the risk of mutation of thisgene, which otherwise may be completely or partially inactivated or giverise to misinformation. It further avoids other risks of using DNA as anagent to induce an immune response (e.g. as a vaccine) such as theinduction of pathogenic anti-DNA antibodies in the patient into whom theforeign DNA has been introduced, so bringing about a (possibly fatal)immune response. In contrast, no anti-RNA antibodies have yet beendetected.

FIGURES

The following Figures are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

FIG. 1: depicts a RNA sequence (SEQ ID NO: 1) (starting sequence basedon the wildtype) encoding MUC1 (HsMUC1—5xVNTR (The wildtype sequencenormally shows 40 tandem repeats. These were—for cloning reasons—reducedto 5 tandem repeats). GC content: 61.27%; length: 1668 bp).

FIG. 2: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 2) encodingMUC1 (HsMUC1 GC—5xVNTR, 1. GC maximized, 2. Codon usage) GC content:73.56%; length 1668 bp. Difference to basic sequence (FIG. 1 (SEQ ID NO:1)): 398/1668 bases=23.86%.

FIG. 3: depicts a RNA sequence (SEQ ID NO: 3) (starting sequence basedon the wildtype) encoding 5T4 (Hs5T4 (trophoblast glycoprotein TPBG); GCcontent: 61.60%; length: 1263 bp.

FIG. 4: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 4) encoding5T4 (Hs5T4 GC, 1. GC-maximized, 2. Codon usage); GC content: 70.47%;length 1263 bp. Difference to basic sequence (FIG. 3 (SEQ ID NO: 3)):247/1263 Bases=19.56%.

FIG. 5: depicts a RNA sequence (SEQ ID NO: 5) (starting sequence basedon the wildtype) encoding Her-2/neu (HsHer2/neu (v-erb-b2 erythroblasticleukemia viral oncogene homolog 2)); GC content: 60.78% ; length: 3768bp.

FIG. 6: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 6) encodingHer-2/neu (HsHer2/neu GC, 1. GC-maximized, 2. Codon usage); GC content:70.54%; length 3768 bp. Difference to basic sequence (FIG. 5 (SEQ ID NO:5)): 772/3768 Bases=20.49%.

FIG. 7: depicts a RNA sequence (SEQ ID NO: 7) (starting sequence basedon the wildtype) encoding hTERT (HsTERT (telomerase reversetranscriptase); GC Content: 66.08%; Length: 3399 bp.

FIG. 8: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 8) encodinghTERT (HsTERT GC, 1. GC-maximized, 2. Codon usage); GC Content: 72.96%;Length 3399 bp, Difference to basic sequence (FIG. 7 (SEQ ID NO: 7)):566/3399 Bases=16.65%.

FIG. 9: depicts a RNA sequence (SEQ ID NO: 9) (starting sequence basedon the wildtype) encoding WT1 (HsWT1 (Wilms tumor 1)); GC Content:61.78%; Length: 1554 bp.

FIG. 10: FIG. 10A) depicts a RNA sequence (SEQ ID NO: 10) encoding WT1(HsWT1 (Wilms tumor 1)) showing a sequence with a reduced GC content inregion 325-408 of said sequence compared to the corresponding region ofthe wildtype sequence.

FIGS. 10B), C) and D) show a comparison of the corresponding regions325-408:

-   -   in B) the wildtype sequence according to FIG. 9 (SEQ ID NO: 9),    -   in C) the GC-maximized sequence according to FIG. 11 (SEQ ID NO:        11), and    -   in D) the GC-reduced sequence according to FIG. 10 (SEQ ID NO:        10), which all show a different GC-pattern.

FIG. 11: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 11) encodingWT1 (HsWT1 GC, 1. GC-maximized, 2. Codon usage); GC Content: 72.59%;Length 1554 bp. Difference to basic sequence (FIG. 9 (SEQ ID NO: 9)):322/1554 Bases=20.72%.

FIG. 12: depicts a RNA sequence (SEQ ID NO: 12) (starting sequence basedon the wildtype) encoding CEA (CEA (carcinoembryonic antigen)HsCEACAM5); GC Content: 52.20%; Length: 2109 bp.

FIG. 13: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 13) encodingCEA (CEACAM5 GC, 1. GC-maximized, 2. Codon usage, already in place); GCContent: 66.24%; Length 2109 bp. Difference to basic sequence (FIG. 12(SEQ ID NO: 12)): 495/2109 Bases=23.47%.

FIG. 14: depicts a RNA sequence (SEQ ID NO: 14) (starting sequence basedon the wildtype) encoding MAGE-A2 (HsMAGE-A2 (melanoma antigen family A,2) HsMAGE-A2B). GC Content: 55.87%; Length: 945 bp.

FIG. 15: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 15) encodingMAGE-A2 (HsMAGE-A2B GC, 1. GC-maximized, 2. Codon usage); GC Content:68.57%; Length 945 bp. Difference to basic sequence (FIG. 14 (SEQ ID NO:14)): 187/945 Bases=19.79%.

FIG. 16: depicts a RNA sequence (SEQ ID NO: 16) (starting sequence basedon the wildtype) encoding MAGE-A3 (MAGE-A3 (melanoma antigen family A,3) MAGE-A3) GC Content: 56.30%; Length: 945 bp.

FIG. 17: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 17) encodingMAGE-A3 (MAGE-A3 GC, 1.GC-maximized, 2. Codon usage, already knownGC-Enrichment); GC Content: 69.00%; Length 945 bp. Difference to basicsequence (FIG. 16 (SEQ ID NO: 16)): 190/945 Bases=20.11%.

FIG. 18: depicts a RNA sequence (SEQ ID NO: 18) (starting sequence basedon the wildtype) encoding Survivin (Survivin (baculoviral IAPrepeat-containing 5, BIRC5) HsSurvivin(wt)); GC Content: 52.68%; Length:429 bp.

FIG. 19: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 19) encodingSurvivin (HsSurvivin(GC), 1. GC-maximized, 2. Codon Usage, already knownGC-Enrichment); GC Content: 65.27%; Length: 429 bp. Difference to basicsequence (FIG. 18 (SEQ ID NO: 18)): 72/429 Bases=16.78%.

FIG. 20: depicts a RNA sequence (SEQ ID NO: 20) (starting sequence basedon the wildtype) encoding NY-ESO-1 (Homo sapiens NY-ESO-1(NY-ESO-1(wt)); GC-Content 67.4%.

FIG. 21: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 21) encodingNY-ESO-1 (NY-ESO-1(GC), GC-Content 79.56%, (already knownGC-Enrichment); Difference to wt (FIG. 20 (SEQ ID NO: 20)): 112/543Bases, 20.63%.

FIG. 22: depicts a RNA sequence (SEQ ID NO: 22) (starting sequence basedon the wildtype) encoding MAGE-C1 (HsMAGEC1 (melanoma antigen familyC, 1) HsMAGEC1(wt)) GC Content: 51.86%; Length: 3429 bp.

FIG. 23: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 23) encodingMAGE-C1 (HsMAGEC1(GC), 1. GC-maximized, 2. Codon usage). GC Content:68.73%; Length 3429 bp. Difference to basic sequence (FIG. 22 (SEQ IDNO: 22)): 964/3429 Bases=28.11%

FIG. 24: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 24) encodinga truncated MAGE-C1 (HsMAGEC1(GC), 1. GC-maximized, 2. Codon usage). Incomparison to the basic sequence (FIG. 22 (SEQ ID NO: 22)) the repeatregions were deleted and the sequence according to FIG. 24, following aninitial start codon (ATG), starts at aa 613 of the GC-maximized wildtypesequence (FIG. 23 (SEQ ID NO: 23)).

FIG. 25: depicts a RNA sequence (SEQ ID NO: 25) (starting sequence basedon the wildtype) encoding MAGE-C2 (HsMAGE-C2 (melanoma antigen family C,2)HsMAGE-C2); GC Content: 50.81%; Length: 1122 bp.

FIG. 26: depicts a (GC) stabilized RNA sequence (SEQ ID NO: 26) encodingMAGE-C2 (HsMAGE-C2 GC, 1. GC-maximized, 2. Codon usage); GC Content:66.58%; Length 1122 bp, Difference to basic sequence (FIG. 25 (SEQ IDNO: 25)): 264/1122 Bases=23.53%.

FIG. 27 shows the presence of IgG1 antibodies specific for the tumourantigen NY-ESO-1 in mice which were vaccinated with the mRNA vaccineconsisting of 5 components, each containing mRNA coding for one NSCLCrelated antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4)formulated with protamine at a mass ratio of 4:1.

FIG. 28: shows the presence of IgG2a antibodies specific for the tumourantigen NY-ESO-1 in mice which were vaccinated with the mRNA vaccineconsisting of 5 components, each containing mRNA coding for one NSCLCrelated antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4)formulated with protamine at a mass ratio of 4:1.

FIG. 29: shows the presence of IgG1 antibodies specific for the tumourantigen MAGE-C1 in mice which were vaccinated with the mRNA vaccineconsisting of 5 components, each containing mRNA coding for one NSCLCrelated antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4)formulated with protamine at a mass ratio of 4:1.

FIG. 30: shows the presence of IgG2a antibodies specific for the tumourantigen MAGE-C1 in mice which were vaccinated with the mRNA vaccineconsisting of 5 components, each containing mRNA coding for one NSCLCrelated antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4)formulated with protamine at a mass ratio of 4:1.

FIG. 31 shows the presence of IgG1 antibodies specific for the tumourantigen MAGE-C2 in mice which were vaccinated with the mRNA vaccineconsisting of 5 components, each containing mRNA coding for one NSCLCrelated antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4)formulated with protamine at a mass ratio of 4:1.

FIG. 32: shows the presence of IgG2a antibodies specific for the tumourantigen MAGE-C2 in mice which were vaccinated with the mRNA vaccineconsisting of 5 components, each containing mRNA coding for one NSCLCrelated antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4)formulated with protamine at a mass ratio of 4:1.

FIG. 33: shows the induction of antigen-specific T-lymphocytes directedagainst the tumour antigen 5T4 in mice which were vaccinated with themRNA vaccine consisting of 5 components, each containing mRNA coding forone NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4)formulated with protamine at a mass ratio of 4:1.

FIG. 34: shows the induction of antigen-specific T-lymphocytes directedagainst the tumour antigen NY-ESO-1 in mice which were vaccinated withthe mRNA vaccine consisting of 5 components, each containing mRNA codingfor one NSCLC related antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and5T4) formulated with protamine at a mass ratio of 4:1.

EXAMPLES

The following examples are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

1. Preparation of Encoding Plasmids:

In the following experiment DNA sequences, corresponding to therespective mRNA sequences end encoding the antigens

-   -   hTERT,    -   WT1,    -   MAGE-A2,    -   5T4,    -   MAGE-A3,    -   MUC1,    -   Her-2/neu,    -   NY-ESO-1,    -   CEA,    -   Survivin,    -   MAGE-C1, or    -   MAGE-C2.

respectively, were prepared and used for in vitro transcription andtransfection experiments. Thereby, the DNA sequence corresponding to thenative antigen encoding mRNA was increased in GC-content andcodon-optimized. Then, the coding sequence was transferred into anRNActive construct (CureVac GmbH, Tubingen, Germany), which has beenmodified with a poly-A-tag and a poly-C-tag (A70-C30).

2. In Vitro Transcription:

Based on the recombinant plasmid DNA obtained in Example 1 the RNAsequences were prepared by in vitro transcription. Therefore, therecombinant plasmid DNA was linearized and subsequently in vitrotranscribed using the T7 RNA polymerase. The DNA template was thendegraded by DNase I digestion, and the RNA was recovered by LiClprecipitation and further cleaned by HPLC extraction (PUREMessenger®,CureVac GmbH, Tubingen, Germany).

3. Complexation With Protamine

For transfection of the RNA into cells and organisms the RNA obtained byin vitro transcription was preferably complexed, more preferably withprotamine upon mixing the RNA with protamine.

4. Vaccination Experiments

For vaccination the RNA obtained by the in vitro transcriptionexperiment as shown above (see Experiment 2) was transfected into mice(Mice: C57 BL/6), preferably when complexed with protamine (seeExperiment 3). Transfection occurred in different groups, wherein 5 mice(C57 BL/6) per group were immunized intradermally 8 times within 3 weekswith the inventive mRNA cocktail, i.e. a mixture of mRNA complexed withprotamine, wherein the RNA codes for at least two of the antigens hTERT,WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin,MAGE-C1, or MAGE-C2.

5. Detection of an Antigen-Specific Immune Response (B-Cell ImmuneResponse):

Detection of an antigen-specific immune response (B-cell immuneresponse) was carried out by detecting antigen-specific antibodies.Therefore, blood samples were taken from the vaccinated mice one weekafter the last vaccination and sera were prepared. MaxiSorb plates(Nalgene Nunc International) were coated with the antigenic protein asencoded by the mRNA-Cocktail (0.5 μg/well). After blocking with 1× PBScontaining 0.05% Tween-20 and 1% BSA the plates were incubated withdiluted mouse serum (1:30, 1:90, 1:270, 1:810). Subsequently abiotin-coupled secondary antibody (Anti-mouse-IgG2a Pharmingen) wasadded. After washing, the plate was incubated with Horseradishperoxidase-streptavidin and subsequently the conversion of the ABTSsubstrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) wasmeasured.

6. Detection of an Antigen-Specific Cellular Immune Response (T CellImmune Response) by ELISPOT:

2 weeks after the last vaccination mice were sacrificed, the spleenswere removed and the splenocytes were isolated. The splenocytes wererestimulated for 7 days in the presence of peptides from the aboveantigens (peptide library) or coincubated with dendritic cells generatedfrom bone marrow cells of native syngeneic mice, which areelectroporated with RNA coding for the antigen. To determine anantigen-specific cellular immune response INFgamma secretion wasmeasured after re-stimulation. For detection of INFgamma a coatmultiscreen plate (Millipore) was incubated overnight with coatingbuffer 0.1 M carbonate-bicarbonate buffer pH 9.6, 10.59 g/l Na₂CO₃, 8.4g/l NaHCO₃) comprising antibody against INFγ (BD Pharmingen, Heidelberg,Germany). Stimulators and effector cells were incubated together in theplate in the ratio of 1:20 for 24 h. The plate was washed with 1× PBSand incubated with a biotin-coupled secondary antibody. After washingwith 1×PBS/0.05% Tween-20 the substrate (5-Bromo-4-Cloro-3-IndolylPhosphate/Nitro Blue Tetrazolium Liquid Substrate System from SigmaAldrich, Taufkirchen, Germany) was added to the plate and the conversionof the substrate could be detected visually.

7. Tumor Challenge:

Immunization:

One week after the last immunization 1 Mio B16 melanoma cells orTRAMP-C1 cells were injected subcutaneously in the mice. Within 2 weeks(B16) or 7 weeks (TRAMP-C1), respectively, tumour volume was determined

8. Preparation of a mRNA Vaccine

A particular example of the inventive active (immunostimulatory)composition, comprising a combination of several antigens for the use asa vaccine for the treatment of non-small cell lung cancer (NSCLC) wasprepared in the following according to the above disclosure. Theexemplary inventive active (immunostimulatory) composition consisted of5 components, each containing mRNA coding for one NSCLC related antigen(NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4, according to SEQ ID NOs:4, 19, 21, 24 and 26 (GC-enriched sequences)) formulated with protamineat a mass ratio of 4:1.

Vaccination

C57BU6 mice were vaccinated intradermally with the mRNA vaccineconsisting of 5 components, each containing mRNA coding for one NSCLCrelated antigen (NY-ESO-1, MAGE-C1, MAGE-C2, Survivin and 5T4, accordingto SEQ ID NOs: 4, 19, 21, 24 and 26 (GC-enriched sequences)) formulatedwith protamine (64 μg per antigen per cycle, divided into 4injections/cycle). Control vaccination was performed using thecorresponding total doses of RNA coding for LacZ (control mRNA lacZ).The vaccination comprised three immunization cycles (week 1, 3, and 5).The groups, number of mice and mouse strains are indicated in thefollowing table:

Groups Mouse strain Number of mice mRNA vaccine C57BL/6 10 5 for Elispotand 5 for antibody detection in serum by ELISA Control mRNA lacZ C57BL/6 5 3 for Elispot and all 5 for antibody detection in serum by ELISA

Detection of Antigen-Specific Antibodies

6 days after last vaccination blood samples (200 μl) were takenretro-orbitally and serum was analyzed for the presence of antigenspecific antibody subtypes IgG1 and IgG2a using ELISA. 96-well ELISAplates were coated with recombinant protein (10 μg/ml in coating buffer,incubation at 37° C. for 4 h) and blocked with 200 μl blocking bufferper well over night at 4° C. Subsequently, the samples were incubatedwith serum pooled from each group of mice and titrated in dilutionsranging from 1:3 to 1:48 for 4 hours at room temperature. Afterincubation with a specific antibody (1:300 in blocking buffer) againstmouse IgG1 or IgG2a and incubation with a HRP-coupled secondary antibody(1:500 in blocking buffer), TMB-substrate was added. The colorimetricreaction was measured at 450 nm using an ELISA reader (Tecan DeutschlandGmbH, Crailsheim, Germany).

ELISPOT

For the detection of cytotoxic T-lymphocyte (CTL) responses the analysisof the secretion of the effector cytokine IFN-γ in response to aspecific stimulus can be visualized at a single cell level using theELISPOT technique.

Splenocytes from antigen-vaccinated and control mice were isolated 6days after last vaccination and then transferred into 96-well ELISPOTplates coated with an anti-IFN-γ capture antibody (10 μg/ml). The cellswere then stimulated for 24 hours at 37° C. either with relevantantigen-derived peptide library or with the HIV-derived library or thesolvent of the peptides, DMSO, or incubated in pure medium as a control.All libraries were used at a concentration of 1 μg/peptide/ml. After theincubation period the cells were washed out of the plate and the IFN-γsecreted by the cells was detected using a biotinylated secondaryantibody against murine IFN-γ (1 μg/ml), followed by streptavidin-AKP.Spots were visualized using BCIP/NBT substrate and counted using anautomated ELISPOT reader (Immunospot Analyzer, CTL Analyzers LLC).

Statistical Analysis

Statistical analysis was performed using Graph Pad Prism 5.01 (GraphPadSoftware, Inc.). All results were expressed as the mean (ormedian)±standard error of means. For Elispot assays, due to the factthat the basal activation is strongly individual dependent, a backgroundcorrection was performed individually per mouse by subtraction of thenumber of spots in medium wells from all other values. Two-tailedMann-Whitney tests were used to analyze difference between the testgroups with a significance level of 5%.

Results and Discussion

Mice were vaccinated with the mRNA vaccine containing five components asdefined above, particularly GC-enriched mRNAs coding for theNSCLC-associated antigens NY-ESO-1, MAGE-C2, MAGE-C1, Survivin and 5T4,(according to SEQ ID NOs: 4, 19, 21, 24 and 26 (GC-enriched sequences))each formulated separately with the cationic peptide protamine at a massratio of 4:1. Control mice were treated with irrelevant RNA coding forLacZ formulated with protamine at the same ratio as the mRNA vaccine.

Using serum isolated from blood drawn from the antigen-vaccinated andcontrol mice, we tested the induction of specific antibodies against theantigens. For three of the five analyzed proteins, MAGE-C1, MAGE-C2 andNY-ESO-1, we detected antigen specific antibodies in serum of micevaccinated with the mRNA vaccine demonstrating that the mRNAs arefunctional and immunogenic in vivo. Proteins required for detection ofantibodies were produced in E. coli. As production of proteins in E.colican influence post-translational modifications and these are not welldescribed for the used antigens, this could account for the lack ofresponse seen for the remaining proteins.

Next the activation of cytotoxic T-cells in response to theadministration of the mRNA vaccine was analyzed. IFN-γ is the mainmediator of Th1 responses and secreted by activated CTLs. Therefore thepresence of antigen-specific cytotoxic T-cells in splenocytes fromvaccinated mice was investigated using the ELISPOT technique. As anantigenic stimulus for splenocytes restricted peptide libraries wereused. Because distinct epitopes of the used human antigens for mouse MHC(H-2K^(b) and H-2D^(b) in C57BL/6 mice) are not known, we had to use ahypothetical selection of peptides selected due to potential bindingaffinity by search of the SYFPEITHI database. Out of peptide libraries(15 mers with 11 amino acids overlap) spanning the whole sequences ofthe proteins, those 15 mers containing the hypothetically best epitopeswere selected and pooled up to a maximum of 18 peptides. However, theseselections might not necessarily contain the correct epitopes so thatthe detection of immune responses with the help of these tools caneasily yield false negative results. Nevertheless, the stimulation withtwo of these libraries, originating from NY-ESO-1 and 5T4, led to highIFN-γ secretion in splenocytes from mice vaccinated with the mRNAvaccine and not in splenocytes from control mice, vaccinated with mRNAcoding for irrelevant protein β-galctosidase. None of the splenocytesreacted to the HIV-derived control peptide library. The number of IFN-γspots by splenocytes incubated in medium alone represents the basalactivation of the freshly isolated cells. Due to the fact that the basalactivation is strongly individual dependent, the background correctionwas performed individually by subtraction of the number of spots inmedium wells from all other values.

The results of these experiments are shown in FIGS. 27 to 34.

1. Active (immunostimulatory) composition comprising at least one RNAencoding at least two different antigens, a) wherein at least one ofthese at least two antigens is (are) selected from: NY-ESO-1, MAGE-C1,and/or MAGE-C2; and b) wherein the further antigen(s) is (are) selectedfrom at least one antigen selected from the following group: hTERT, WT1,MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin,MAGE-C1, and/or MAGE-C2.
 2. Active (immunostimulatory) compositionaccording to claim 1, wherein at least one of these at least twodifferent antigens according to a) is selected from MAGE-C1, and/orMAGE-C2.
 3. Active (immunostimulatory) composition according to claim 1,wherein the further antigen(s) according to b) is (are) exclusivelyselected from the following combinations of antigens: hTERT and WT1; orhTERT and MAGE-A2; or hTERT and 5T4; or hTERT and MAGE-A3; or hTERT andMUC1; or hTERT and Her-2/neu; or hTERT and NY-ESO-1; or hTERT and CEA;or hTERT and Survivin; or hTERT and MAGE-C1; or hTERT and MAGE-C2; orWT1 and MAGE-A2; or WT1 and 5T4; or WT1 and MAGE-A3; or WT1 and MUC1; orWT1 and Her-2/neu; or WT1 and NY-ESO-1; or WT1 and CEA; or WT1 andSurvivin; or WT1 and MAGE-C1; or WT1 and MAGE-C2; or MAGE-A2 and 5T4; orMAGE-A2 and MAGE-A3; or MAGE-A2 and MUC1 ; or MAGE-A2 and Her-2/neu; orMAGE-A2 and NY-ESO-1; or MAGE-A2 and CEA; or MAGE-A2 and Survivin; orMAGE-A2 and MAGE-C1; or MAGE-A2 and MAGE-C2; or 5T4 and MAGE-A3; or 5T4and MUC1; or 5T4 and Her-2/neu; or 5T4 and NY-ESO-1; or 5T4 and CEA; or5T4 and Survivin; or 5T4 and MAGE-C1; or 5T4 and MAGE-C2; or MAGE-A3 andMUC1; or MAGE-A3 and Her-2/neu; or MAGE-A3 and NY-ESO-1; or MAGE-A3 andCEA; or MAGE-A3 and Survivin; or MAGE-A3 and MAGE-C1 MAGE-A3 and MAGE-C2MUC1 and Her-2/neu; or MUC1 and NY-ESO-1; or MUC1 and CEA; or MUC1 andSurvivin; or MUC1 and MAGE-C1; or MUC1 and MAGE-C2; or HER-2/NEU andNY-ESO-1; or HER-2/NEU and CEA; or HER-2NEU and Survivin; or HER-2/NEUand MAGE-C1; or HER-2/NEU and MAGE-C2; or NY-ESO-1 and CEA; or NY-ESO-1and Survivin; or NY-ESO-1 and MAGE-C1 ; or NY-ESO-1 and MAGE-C2; or CEAand Survivin; or CEA and MAGE-C1; or CEA and MAGE-C2; or Survivin andMAGE-C1; or Survivin and MAGE-C2; or MAGE-C1 and MAGE-C2; or hTERT, WT1and MAGE-A2; or hTERT, WT1 and 5T4; or hTERT, WT1 and MAGE-A3; or hTERT,WT1 and MUC1; or hTERT, WT1 and Her-2/neu; or hTERT, WT1 and NY-ESO-1;or hTERT, WT1 and CEA; or hTERT, WT1 and Survivin; or hTERT, WT1 andMAGE-C1; or hTERT, WT1 and MAGE-C2; or WT1, MAGE-A2 and 5T4; or WT1,MAGE-A2 and MAGE-A3; or WT1, MAGE-A2 and MUC1; or WT1, MAGE-A2 andHer-2/neu; or WT1, MAGE-A2 and NY-ESO-1; or WT1, MAGE-A2 and CEA; orWT1, MAGE-A2 and Survivin; or WT1, MAGE-A2 and MAGE-C1; or WT1, MAGE-A2and MAGE-C2; or MAGE-A2, 5T4 and MAGE-A3; or MAGE-A2, 5T4 and MUC1; orMAGE-A2, 5T4 and Her-2/neu; or MAGE-A2, 5T4 and NY-ESO-1; or MAGE-A2,5T4 and CEA; or MAGE-A2, 5T4 and Survivin; or MAGE-A2, 5T4 and MAGE-C1;or MAGE-A2, 5T4 and MAGE-C2; or 5T4, MAGE-A3 and MUC1; or 5T4, MAGE-A3and Her-2/neu; or 5T4, MAGE-A3 and NY-ESO-1; or 5T4, MAGE-A3 and CEA; or5T4, MAGE-A3 and Survivin; or 5T4, MAGE-A3 and MAGE-C1; or 5T4, MAGE-A3and MAGE-C2; or MAGE-A3, MUC1 and Her-2/neu; or MAGE-A3, MUC1 andNY-ESO-1; or MAGE-A3, MUC1 and CEA; or MAGE-A3, MUC1 and Survivin; orMAGE-A3, MUC1 and MAGE-C1; or MAGE-A3, MUC1 and MAGE-C2; or MUC1,Her-2/neu and NY-ESO-1; or MUC1, Her-2/neu and CEA; or MUC1, Her-2/neuand Survivin; or MUC1, Her-2/neu and MAGE-C1; or MUC1, Her-2/neu andMAGE-C2; or HER-2/NEU, NY-ESO-1 and CEA; or HER-2/NEU, NY-ESO-1 andSurvivin; or HER-2/NEU, NY-ESO-1 and MAGE-C1; or HER-2/NEU, NY-ESO-1 andMAGE-C2; or NY-ESO-1, CEA and Survivin; or NY-ESO-1, CEA and MAGE-C1; orNY-ESO-1, CEA and MAGE-C2; or CEA, Survivin and MAGE-C1; or CEA,Survivin and MAGE-C2; or Survivin, MAGE-C1 and MAGE-C2; or hTERT, WT1,MAGE-A2 and 5T4; or hTERT, WT1, MAGE-A2 and MAGE-A3; or hTERT, WT1,MAGE-A2 and MUC1 ; or hTERT, WT1, MAGE-A2 and Her-2/neu; or hTERT, WT1,MAGE-A2 and NY-ESO-1; or hTERT, WT1, MAGE-A2 and CEA; or hTERT, WT1,MAGE-A2 and Survivin; or hTERT, WT1, MAGE-A2 and MAGE-C1; or hTERT, WT1,MAGE-A2 and MAGE-C2; or WT1, MAGE-A2, 5T4 and MAGE-A3; or WT1, MAGE-A2,5T4 and MUC1; or WT1, MAGE-A2, 5T4 and Her-2/neu; or WT1, MAGE-A2, 5T4and NY-ESO-1; or WT1, MAGE-A2, 5T4 and CEA; or WT1, MAGE-A2, 5T4 andSurvivin; or WT1, MAGE-A2, 5T4 and MAGE-C1; or WT1, MAGE-A2, 5T4 andMAGE-C2; or MAGE-A2, 5T4, MAGE-A3 and MUC1; or MAGE-A2, 5T4, MAGE-A3 andHer-2/neu; or MAGE-A2, 5T4, MAGE-A3 and NY-ESO-1; or MAGE-A2, 5T4,MAGE-A3 and CEA; or MAGE-A2, 5T4, MAGE-A3 and Survivin; or MAGE-A2, 5T4,MAGE-A3 and MAGE-C1; or MAGE-A2, 5T4, MAGE-A3 and MAGE-C2; or 5T4,MAGE-A3, MUC1, and Her-2/neu; or 5T4, MAGE-A3, MUC1 and NY-ESO-1; or5T4, MAGE-A3, MUC1 and CEA; or 5T4, MAGE-A3, MUC1 and Survivin; or 5T4,MAGE-A3, MUC1 and MAGE-C1; or 5T4, MAGE-A3, MUC1 and MAGE-C2; orMAGE-A3, MUC1, Her-2/neu and NY-ESO-1; or MAGE-A3, MUC1, Her-2/neu andCEA; or MAGE-A3, MUC1, Her-2/neu and Survivin; or MAGE-A3, MUC1,Her-2/neu and MAGE-C1; or MAGE-A3, MUC1, Her-2/neu and MAGE-C2; or MUC1,Her-2/neu, NY-ESO-1 and CEA; or MUC1, Her-2/neu, NY-ESO-1 and Survivin;or MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1 ; or MUC1, Her-2/neu, NY-ESO-1and MAGE-C2; or HER-2/NEU, NY-ESO-1, CEA and Survivin; or HER-2/NEU,NY-ESO-1, CEA and MAGE-C1; or HER-2/NEU, NY-ESO-1, CEA and MAGE-C2; orNY-ESO-1, CEA, Survivin and MAGE-C1; or NY-ESO-1, CEA, Survivin andMAGE-C2; or CEA, Survivin, MAGE-C1 and MAGE-C2; or hTERT, WT1, MAGE-A2,5T4 and MAGE-A3; or hTERT, WT1, MAGE-A2, 5T4 and MUC1; or hTERT, WT1,MAGE-A2, 5T4 and Her-2/neu; or hTERT, WT1, MAGE-A2, 5T4 and NY-ESO-1; orhTERT, WT1, MAGE-A2, 5T4 and CEA; or hTERT, WT1, MAGE-A2, 5T4 andSurvivin; or hTERT, WT1, MAGE-A2, 5T4 and MAGE-C1; or hTERT, WT1,MAGE-A2, 5T4 and MAGE-C2; or WT1, MAGE-A2, 5T4, MAGE-A3 and MUC1; orWT1, MAGE-A2, 5T4, MAGE-A3 and Her-2/neu; or WT1, MAGE-A2, 5T4, MAGE-A3and NY-ESO-1; or WT1, MAGE-A2, 5T4, MAGE-A3 and CEA; or WT1, MAGE-A2,5T4, MAGE-A3 and Survivin; or WT1, MAGE-A2, 5T4, MAGE-A3 and MAGE-C1; orWT1, MAGE-A2, 5T4, MAGE-A3 and MAGE-C2; or MAGE-A2, 5T4, MAGE-A3, MUC1and Her-2/neu; or MAGE-A2, 5T4, MAGE-A3, MUC1 and NY-ESO-1; or MAGE-A2,5T4, MAGE-A3, MUC1 and CEA; or MAGE-A2, 5T4, MAGE-A3, MUC1 and Survivin;or MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C1; or MAGE-A2, 5T4, MAGE-A3,MUC1 and MAGE-C2; or 5T4, MAGE-A3, MUC1, Her-2/neu and NY-ESO-1; or 5T4,MAGE-A3, MUC1, Her-2/neu and CEA; or 5T4, MAGE-A3, MUC1, Her-2/neu andSurvivin; or 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C1; or 5T4, MAGE-A3,MUC1, Her-2/neu and MAGE-C2; or MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 andCEA; or MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and Survivin; or MAGE-A3,MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1; or MAGE-A3, MUC1, Her-2/neu,NY-ESO-1 and MAGE-C2; or MUC1, Her-2/neu, NY-ESO-1, CEA and Survivin; orMUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1; or MUC1, Her-2/neu,NY-ESO-1, CEA and MAGE-C2; or HER-2/NEU, NY-ESO-1, CEA, Survivin andMAGE-C1; or HER-2/NEU, NY-ESO-1, CEA, Survivin and MAGE-C2; or NY-ESO-1,CEA, Survivin, MAGE-C1 and MAGE-C2; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3and MUC1; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and Her-2/neu; or hTERT,WT1, MAGE-A2, 5T4, MAGE-A3 and NY-ESO-1; or hTERT, WT1, MAGE-A2, 5T4,MAGE-A3 and CEA; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and Survivin; orhTERT, WT1, MAGE-A2, 5T4, MAGE-A3 and MAGE-C1; or hTERT, WT1, MAGE-A2,5T4, MAGE-A3 and MAGE-C2; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 andHer-2/neu; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and NY-ESO-1; or WT1,MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1and Survivin; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C1; or WT1,MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C2; or MAGE-A2, 5T4, MAGE-A3, MUC1,Her-2/neu and NY-ESO-1; or MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu andCEA; or MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and Survivin; or MAGE-A2,5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C1; or MAGE-A2, 5T4, MAGE-A3,MUC1, Her-2/neu and MAGE-C2; or 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1and CEA; or 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and Survivin; or5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1; or 5T4, MAGE-A3,MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2; or MAGE-A3, MUC1, Her-2/neu,NY-ESO-1, CEA and Survivin; or MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEAand MAGE-C1 ; or MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2; orMUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C1; or MUC1,Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C2; or HER-2/NEU, NY-ESO-1,CEA, Survivin, MAGE-C1 and MAGE-C2; or hTERT, WT1, MAGE-A2, 5T4,MAGE-A3, MUC1 and Her-2/neu; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1and NY-ESO-1; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and CEA; orhTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1 and Survivin; or hTERT, WT1,MAGE-A2, 5T4, MAGE-A3, MUC1 and MAGE-C1; or hTERT, WT1, MAGE-A2, 5T4,MAGE-A3, MUC1 and MAGE-C2; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1,Her-2/neu and NY-ESO-1; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neuand CEA; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and Survivin; orWT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C1; or WT1,MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C2; or MAGE-A2, 5T4,MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and CEA; or MAGE-A2, 5T4, MAGE-A3,MUC1, Her-2/neu, NY-ESO-1 and Survivin; or MAGE-A2, 5T4, MAGE-A3, MUC1,Her-2/neu, NY-ESO-1 and MAGE-C1; or MAGE-A2, 5T4, MAGE-A3, MUC1,Her-2/neu, NY-ESO-1 and MAGE-C2; or 5T4, MAGE-A3, MUC1, Her-2/neu,NY-ESO-1, CEA and Survivin; or 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1,CEA and MAGE-C1; or 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA andMAGE-C2; or MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin andMAGE-C1; or MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin andMAGE-C2; or MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1 andMAGE-C2; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu andNY-ESO-1; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and CEA;or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and Survivin; orhTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C1; orhTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu and MAGE-C2; or WT1,MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and CEA; or WT1,MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and Survivin; or WT1,MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C1; or WT1,MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and MAGE-C2, orMAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and Survivin; orMAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1; orMAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2; or5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C1; or5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C2; orMAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1 and MAGE-C2;or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 and CEA;or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1 andSurvivin; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu,NY-ESO-1 and MAGE-C1; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1,Her-2/neu, NY-ESO-1 and MAGE-C2; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1,Her-2/neu, NY-ESO-1, CEA and Survivin; or WT1, MAGE-A2, 5T4, MAGE-A3,MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1; or WT1, MAGE-A2, 5T4,MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2; or MAGE-A2, 5T4,MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C1; orMAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin andMAGE-C2; or 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin,MAGE-C1 and MAGE-C2; or hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1,Her-2/neu, NY-ESO-1, CEA and Survivin; or hTERT, WT1, MAGE-A2, 5T4,MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C1; or hTERT, WT1,MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA and MAGE-C2; orWT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin andMAGE-C1; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA,Survivin and MAGE-C2; or MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu,NY-ESO-1, CEA, Survivin, MAGE-C1 and MAGE-C2; or hTERT, WT1, MAGE-A2,5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin and MAGE-C1; orhTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA,Survivin and MAGE-C2; or WT1, MAGE-A2, 5T4, MAGE-A3, MUC1, Her-2/neu,NY-ESO-1, CEA, Survivin, MAGE-C1 and MAGE-C2; or hTERT, WT1, MAGE-A2,5T4, MAGE-A3, MUC1, Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1 andMAGE-C2.
 4. Active (immunostimulatory) composition according to claim 1,wherein the at least one RNA comprises a length of 250 to 20000nucleotides.
 5. Active (immunostimulatory) composition according toclaim 1, wherein the at least one RNA is a mRNA.
 6. Active(immunostimulatory) composition according to claim 1, wherein the atleast one RNA is a monocistronic, bicistronic or even multicistronicRNA.
 7. Active (immunostimulatory) composition according to claim 5,wherein the at least two antigens are each encoded by a monocistronicRNA.
 8. Active (immunostimulatory) composition according to claim 6,wherein the at least two antigens are encoded by a mixture ofmonocistronic, bicistronic and/or even multicistronic RNAs.
 9. Active(immunostimulatory) composition according to claim 1, wherein the atleast one RNA comprises a RNA selected from RNAs encoding a fragment, avariant or an epitope of an antigen as defined in claim
 1. 10. Active(immunostimulatory) composition according to claim 1, wherein the atleast one RNA comprises a RNA selected from RNAs being identical or atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% identical to the RNAsequences according to SEQ ID NOs: 1, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22or
 25. 11. Active (immunostimulatory) composition according to claim 1,wherein the at least one RNA is a modified RNA, in particular astabilized mRNA.
 12. Active (immunostimulatory) composition according toclaim 11, wherein the G/C content of the coding region of the at leastone RNA is increased compared to the G/C content of the coding region ofthe wild-type RNA, the coded amino acid sequence of the at least one RNApreferably not being modified compared to the encoded amino acidsequence of the wild-type RNA.
 13. Active (immunostimulatory)composition according to claim 11, wherein the A/U content in theenvironment of the ribosome binding site of the at least one RNA isincreased compared with the A/U content in the environment of theribosome binding site of the wild-type RNA.
 14. Active(immunostimulatory) composition according to claim 11, wherein thecoding region and/or the 5′ and/or 3′ untranslated region of themodified mRNA is modified compared to the wild-type RNA such that itcontains no destabilizing sequence elements, the coded amino acidsequence of the modified mRNA preferably not being modified compared tothe wild-type RNA.
 15. Active (immunostimulatory) composition accordingto claim 11, wherein the modified mRNA has a 5′ cap structure and/or apoly(A) tail, preferably of 10 to 200 adenosine nucleotides, and/or apoly(C) tail, preferably of 10 to 200 cytosine nucleotides, and/or atleast one IRES and/or at least one 5′ and/or 3′ stabilizing sequence.16. Active (immunostimulatory) composition according to claim 1, whereinthe at least one RNA, preferably all mRNAs, comprises a RNA selectedfrom RNAs being identical or at least 80% identical to the RNA sequencesaccording to SEQ ID NOs: 2, 4, 6, 8, 10, 11, 13, 15, 17, 19, 21, 23, 24or
 26. 17. Active (immunostimulatory) composition according to claim 1,wherein the at least one RNA is complexed with one or more polycations,preferably with protamine or oligofectamine, most preferably withprotamine.
 18. Active (immunostimulatory) composition according to claim1, wherein the active composition additionally comprises at least oneadjuvant.
 19. Active (immunostimulatory) composition according to claim18, wherein the at least one adjuvant is selected from the groupconsisting of: cationic or polycationic compounds, comprising cationicor polycationic peptides or proteins, including protamine, nucleoline,spermin or spermidine, poly-L-lysine (PLL), poly-arginine, basicpolypeptides, cell penetrating peptides (CPPs), including HIV-bindingpeptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA orprotein transduction domains (PTDs, PpT620, prolin-rich peptides,arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1,L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, plsl, FGF,Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC,hCT-derived peptides, SAP, protamine, spermine, spermidine, or histones,cationic polysaccharides, including chitosan, polybrene, cationicpolymers, including polyethyleneimine (PEI), cationic lipids, includingDOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride,DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP,DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC,DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, including modifiedpolyaminoacids, including β-aminoacid-polymers or reversed polyamides,modified polyethylenes, including PVP (poly(N-ethyl-4-vinylpyridiniumbromide)), modified acrylates, including pDMAEMA(poly(dimethylaminoethyl methylacrylate)), modified Amidoaminesincluding pAMAM (poly(amidoamine)), modified polybetaaminoester (PBAE),including diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, dendrimers, includingpolypropylamine dendrimers or pAMAM based dendrimers, polyimine(s),including PEI: poly(ethyleneimine), poly(propyleneimine),polyallylamine, sugar backbone based polymers, including cyclodextrinbased polymers, dextran based polymers, Chitosan, etc., silan backbonebased polymers, such as PMOXA-PDMS copolymers, etc., Blockpolymersconsisting of a combination of one or more cationic blocks selected of acationic polymer as mentioned before, and of one or more hydrophilic- orhydrophobic blocks (e.g polyethyleneglycole); or cationic orpolycationic proteins or peptides, selected from following proteins orpeptides having the following total formula (I):(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), whereinl+m+n+o+x=8-15, and l, m, n or o independently of each other may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide; ornucleic acids having the formula (II): G_(l)X_(m)G_(n), wherein: G isguanosine, uracil or an analogue of guanosine or uracil; X is guanosine,uracil, adenosine, thymidine, cytosine or an analogue of theabove-mentioned nucleotides; 1 is an integer from 1 to 40, wherein whenl=1 G is guanosine or an analogue thereof, when l>1 at least 50% of thenucleotides are guanosine or an analogue thereof; in is an integer andis at least 3; wherein when m=3 X is uracil or an analogue thereof, whenm>3 at least 3 successive uracils or analogues of uracil occur; n is aninteger from 1 to 40, wherein when n=1 G is guanosine or an analoguethereof, when n>1 at least 50% of the nucleotides are guanosine or ananalogue thereof; or nucleic acids having the formula (III):C_(l)X_(m)C_(n), wherein: C is cytosine, uracil or an analogue ofcytosine or uracil; X is guanosine, uracil, adenosine, thymidine,cytosine or an analogue of the above-mentioned nucleotides; 1 is aninteger from 1 to 40, wherein when l=1 C is cytosine or an analoguethereof, when l>1 at least 50% of the nucleotides are cytosine or ananalogue thereof; m is an integer and is at least 3; wherein when m=3 Xis uracil or an analogue thereof, when m>3 at least 3 successive uracilsor analogues of uracil occur; n is an integer from 1 to 40, wherein whenn=1 C is cytosine or an analogue thereof, when n>1 at least 50% of thenucleotides are cytosine or an analogue thereof
 20. Vaccine, comprisingan active (immunostimulatory) composition according to claim
 1. 21.Vaccine according to claim 20, wherein the active (immunostimulatory)composition elicits an adaptive immune response.
 22. Vaccine accordingto claim 20, wherein the vaccine further comprises a pharmaceuticallyacceptable carrier.
 23. Vaccine according to claim 21, wherein thevaccine further comprises a pharmaceutically acceptable carrier.
 24. Useof an active (immunostimulatory) composition according to claim 1 forpreparing a vaccine for the treatment of lung cancer, preferably of anon-small-cell lung cancer (NSCLC) related condition, more preferably ofconditions related to the three main sub-types of non-small-cell lungcancer (NSCLC) including squamous cell lung carcinoma, adenocarcinomaand large cell lung carcinoma.
 25. Kit, preferably kits of parts,comprising the active (immunostimulatory) composition according to claim1, and optionally technical instructions with information on theadministration and dosage of the active (immunostimulatory) compositionand/or the vaccine.